Method for the diagnosis of Gaucher&#39;s disease

ABSTRACT

The present invention is related to an in vitro method for diagnosing Gaucher&#39;s disease in a subject comprising a step ofa) detecting a biomarker in a sample from the subject, wherein the biomarker is free lyso-Gb1.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 14/124,375, filed Mar. 13, 2014, which is a U.S.C. § 371 nationalphase application of PCT International Application No.PCT/EP2012/002409, filed Jun. 6, 2012, which claims priority to EuropeanPatent Application No. 11004597.8, filed Jun. 6, 2011. The disclosuresof each are hereby incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The present invention is related to a method for diagnosing Gaucher'sdisease in a subject, a method for determining the course of Gaucher'sdisease in a subject, a method for determining the effectiveness of atleast one treatment applied to a subject being positively tested forsuffering from or being at risk for developing Gaucher's disease, amethod of determining the effectiveness of a compound for the treatmentof Gaucher's disease, use of mass spectrometry for the detection of abiomarker, use of a biomarker for Gaucher's disease, a kit fordetermining the presence of a biomarker in a sample from a subject and asoftware product, wherein the biomarker is free lyso-Gb1.

BACKGROUND OF THE INVENTION

Lysosomal storage diseases, also referred to herein as lysosomal storagedisorders or LSDs, are a group of rare inherited metabolic disordersthat result from defects in lysosomal function. LSDs result when aspecific organelle in the body's cells—the lysosome—malfunctions. Someof the more prominent lysosomal storage diseases are Gaucher's diseaseand Fabry disease.

LSDs are caused by lysosomal dysfunction usually as a consequence ofdeficiency of a single enzyme required for the metabolism of lipids,glycoproteins or so-called mucopolysaccharides. Individually, LSDs occurwith frequencies of about 1:10,000 to 1:250,000, however, as a group theincidence is about 1:5,000. Most of these disorders are autosomalrecessively inherited; however, a few are X-linked inherited, such asFabry disease and Hunter syndrome (MPS II).

Like other genetic diseases, individuals typically inherit lysosomalstorage diseases from their parents. Although each disorder results fromdifferent gene mutations that translate into a deficiency in enzymeactivity, they all share a common biochemical characteristic—nearly alllysosomal disorders originate from an abnormal accumulation ofsubstances inside the lysosome.

Lysosomal storage diseases affect mostly children and they often die ata young and unpredictable age, many within a few months or years ofbirth. Many other children die of this disease following years ofsuffering from various symptoms of their particular disorder.

The symptoms of lysosomal storage disease vary, depending on theparticular disorder and other variables like the age of onset, and canbe mild to severe. They can include developmental delay, movementdisorders, seizures, dementia, deafness and/or blindness. Some peoplewith Lysosomal storage disease have enlarged livers (hepatomegaly) andenlarged spleens (splenomegaly), pulmonary and cardiac problems, andbones that develop abnormally.

There are no causative cures for lysosomal storage diseases andtreatment is mostly symptomatic, although bone marrow transplantationand enzyme replacement therapy (ERT) have been used for some indicationswith good success. In addition, umbilical cord blood transplantation isbeing performed at specialized centers for a number of these diseases.In addition, substrate reduction therapy (SRT), a method used todecrease the accumulation of storage material, is currently beingevaluated for some of these diseases. Furthermore, chaperone therapy, atechnique used to stabilize the defective enzymes produced by patients,is being examined for certain of these disorders. Gene therapyconstitutes a further option for the treatment of these diseases.

To date a definitive diagnosis of Gaucher's disease can only be madeapplying biochemical testing measuring directly the defect of thebeta-glucosidase enzyme together with genetic confirmation. Sincenumerous different mutations may be the cause of a particular lysosomalstorage disease the sequencing of the entire beta-glucosidase gene isapplied in Gaucher's disease in order to confirm the diagnosis.

Although there are attempts to apply diagnosis methods based onassociated biochemical abnormalities such as high alkaline phosphatase,angiotensin-converting enzyme (ACE) and immunoglobulin levels, or, incase of Gaucher's disease, by cell analysis showing “crinkled paper”cytoplasm and glycolipid-laden macrophages, there is an unmet need for asimple biochemical test exhibiting highly specific and highly sensitivedetection of said lysosomal storage disease at an early stage,monitoring progression of the disease and early monitoring the efficacyof applied therapies.

Therefore, the identification of biomarkers for the early detection anddiagnosis of Gaucher's diseases holds great promise to improve theclinical outcome of patients. It is especially important for patientswith vague or no symptoms or to detect patients which fail to respond toa therapy.

A biomarker should be technically feasible in many hands, easy tomeasure; useful, with a consistent, relative magnitude betweenexperimentals/patients and controls, or treated and untreated; reliable,precise, and accurate clinically, and classifiable as stronglypredictive or prognostic.

In Gaucher's disease some lysosomal enzymes, used as indirectbiomarkers, were found to be elevated, including tartrate-resistant acidphosphatase, hexosaminidase, and a human chitinase, chitotriosidase.Thus there are attempts to monitor the reduction of storage cells intissues by measurement of such surrogate markers of Gaucher cells likechitotriosidase and CCL18 (C. E. Hollak et al. Marked elevation ofplasma chitotriosidase activity. A novel hallmark of Gaucher disease, J.Clin. Invest. 93 (1994) 1288-1292; R. G. Boot et al. Marked elevation ofthe chemokine CCL18/PARC in Gaucher disease: a novel surrogate markerfor assessing therapeutic intervention, Blood 103 (2004) 33-39).However, beside other disadvantages in the use of chitotriosidase as abiomarker for Gaucher's disease, said enzyme accumulates independent ofa direct link to the pathology of Gaucher's disease. Furthermore, up to35% of given ethnicities demonstrate a defect of the gene coding forchitotriosidase resulting in an artificially reduced or non-measurablechitotriosidase activity.

The use of primary storage molecules as biomarker was assessed forglucosyl ceramide (Gb1) in plasma of Gaucher's disease patients andcompared to the level of Gb1 in healthy individuals (Groener et al.Biochim Biophys Acta. 2008 January-February; 1781(1-2):72-8. Epub 2007Dec. 5.; Plasma glucosylceramide and ceramide in type 1 Gaucher diseasepatients: correlations with disease severity and response to therapeuticintervention.; Groener J E et al.). Nevertheless, although Gb1 measuredin said study was increased in plasma of said patients, said increase ofGb1 was not prominent and thus the specificity and the sensitivity ofthe method were low showing that Gb1 is not applicable as a biomarkerfor Gaucher's disease.

Already in 1989 Rosengren et al. (Lysosulfatide(galactosylsphingosine-3-O-sulfate) from metachromatic leukodystrophyand normal human brain, Rosengren B, Fredman P, Månsson J E, SvennerholmL.; J Neurochem. 1989 April; 52(4):1035-41.) showed that in lipidosesnot only the catabolism of the major sphingolipid but also itslyso-compound is affected. Nevertheless, said study concluded that thelyso-compounds do not play a key-role in the pathogenetic mechanisms inthe sphingolipidoses. Thus, said lyso-compounds might not be suitablebiomarkers for diagnosis of sphingolipidoses such as Gaucher's disease.

It is important to note that until today no use of a highly specific andhighly sensitive biomarker and no method for the diagnosis of Gaucher'sdisease is available beside the methods described above, that exhibit anunsatisfactory limit of detection, sensitivity and/or specificity andthus proved to be unsuitable for clinical application.

Accordingly, there is need for a fast, simple and more importantlyreliable method for the diagnosis of Gaucher's disease.

In the light of the above, the problem underlying the present inventionis to provide a method for the diagnosis of Gaucher's disease.

A further problem underlying the present invention is to provide amethod for determining the course and prognosis of Gaucher's disease.

A still further problem underlying the present invention is to provide amethod for determining rather quickly the effectiveness of at least onetreatment applied to a subject being positively tested for sufferingfrom or being at risk of developing Gaucher's disease.

A further problem underlying the present invention is to provide amethod for determining the effectiveness of a compound for the treatmentof a Gaucher's disease.

Another problem underlying the present invention is to provide abiomarker which allows the specific and sensitive diagnosis of Gaucher'sdisease. A still further problem underlying the present invention is akit which comprises a compound which interacts with a biomarker which isspecific and sensitive for Gaucher's disease.

These and other problems are solved by the subject matter of theattached independent claims. Preferred embodiments may be taken from thedependent claims.

The problem underlying the present invention is solved in a first aspectwhich is also the first embodiment of the first aspect, by a method fordiagnosing Gaucher's disease in a subject comprising a step of

-   -   a) detecting a biomarker in a sample from the subject, wherein        the biomarker is free lyso-Gb1.

In a second embodiment of the first aspect which is also an embodimentof the first embodiment of the first aspect, the method furthercomprises a step of

-   -   b) determining a level of the biomarker present in the sample.

In a third embodiment of the first aspect which is also an embodiment ofthe first and the second embodiment of the first aspect, the level ofthe biomarker is indicative whether the subject is suffering from orwhether the subject is at risk for developing Gaucher's disease.

In a fourth embodiment of the first aspect which is also an embodimentof the first, the second and the third embodiment of the first aspect,the sample from the subject is a sample from a subject who has beenpreviously treated or diagnosed for Gaucher's disease.

In a fifth embodiment of the first aspect which is also an embodiment ofthe first, the second and the third embodiment of the first aspect, thesample from the subject is a sample from a subject who has not beenpreviously treated or a subject who has not been previously diagnosedfor Gaucher's disease.

In a sixth embodiment of the first aspect which is also an embodiment ofthe first, the second, the third, the fourth and the fifth embodiment ofthe first aspect, the method further comprises a step of

-   -   c) applying, maintaining, reducing, elevating or not applying a        therapy based on the diagnosis of whether the subject is        suffering from or for being at risk for developing Gaucher's        disease.

In a seventh embodiment of the first aspect which is also an embodimentof the first, the second, the third, the fourth, the fifth and the sixthembodiment of the first aspect, the method further comprises a step of

-   -   d) detecting the biomarker in a sample from the subject after        applying, maintaining, reducing, elevating or not applying a        therapy in a step of c).

In an eighth embodiment of the first aspect which is also an embodimentof the first, the second, the third, the fourth, the fifth, the sixthand the seventh embodiment of the first aspect, the method furthercomprises a step of

-   -   e) determining a level of the biomarker in the sample from the        subject after applying, maintaining, reducing, elevating or not        applying a therapy in a step of c).

In a ninth embodiment of the first aspect which is also an embodiment ofthe eighth embodiment of the first aspect, the method further comprisesthe step of

-   -   f) determining whether the level of the biomarker determined in        step b) is lower than the level of the biomarker determined in        step e).

In a tenth embodiment of the first aspect which is also an embodiment ofthe ninth embodiment of the first aspect, the method further comprisesthe step of

-   -   g) applying, maintaining, reducing, elevating or not applying a        therapy based on the step off).

In an eleventh embodiment of the first aspect which is also anembodiment of the first, the second, the third, the fourth, the fifth,the sixth, the seventh, the eighth, the ninth and the tenth embodimentof the first aspect, the method further comprises detecting at least oneadditional biomarker in the sample from the subject.

In a twelfth embodiment of the first aspect which is also an embodimentof the eleventh embodiment of the first aspect, the method furthercomprises determining the level of the at least one additional biomarkerin the sample from the subject.

In a thirteenth embodiment of the first aspect which is also anembodiment of the eleventh and the twelfth embodiment of the firstaspect, the at least one additional biomarker is selected from the groupcomprising chitotriosidase and CCL18.

In a fourteenth embodiment of the first aspect which is also anembodiment of the thirteenth embodiment of the first aspect, the atleast one additional biomarker is chitotriosidase.

In a fifteenth embodiment of the first aspect which is also anembodiment of the thirteenth embodiment of the first aspect, the atleast one additional biomarker is CCL18.

In a sixteenth embodiment of the first aspect which is also anembodiment of the first, the second, the third, the fourth, the fifth,the sixth, the seventh, the eighth, the ninth, the tenth, the eleventh,the twelfth, the thirteenth, the fourteenth and the fifteenth embodimentof the first aspect, the method further comprises detectingchitotriosidase and CCL18.

In a seventeenth embodiment of the first aspect which is also anembodiment of the first, the second, the third, the fourth, the fifth,the sixth, the seventh, the eighth, the ninth, the tenth, the eleventh,the twelfth, the thirteenth, the fourteenth, the fifteenth and thesixteenth embodiment of the first aspect, the biomarker and/or the atleast one additional biomarker is detected by means of immunoassay, massspectrometric analysis, biochip array, functional nucleic acids and/or afluorescent derivative of free lyso-Gb1.

In an eighteenth embodiment of the first aspect which is also anembodiment of the seventeenth embodiment of the first aspect, thebiomarker is detected by means of mass spectrometric analysis.

In a nineteenth embodiment of the first aspect which is also anembodiment of the eighteenth embodiment of the first aspect, massspectrometric analysis is selected from the group consisting of SELDI,MALDI, MALDI-Q TOF, MS/MS, TOF-TOF and ESI-O-TOF.

In a twentieth embodiment of the first aspect which is also anembodiment of the nineteenth embodiment of the first aspect, the massspectrometric analysis uses MS/MS.

In a twenty first embodiment of the first aspect which is also anembodiment of the first, the second, the third, the fourth, the fifth,the sixth, the seventh, the eighth, the ninth, the tenth, the eleventh,the twelfth, the thirteenth, the fourteenth, the fifteenth, thesixteenth, the seventeenth, the eighteenth, the nineteenth and thetwentieth embodiment of the first aspect, the method further comprisesprotein precipitation and/or HPLC.

In a twenty second embodiment of the first aspect which is also anembodiment of the first, the second, the third, the fourth, the fifth,the sixth, the seventh, the eighth, the ninth, the tenth, the eleventh,the twelfth, the thirteenth, the fourteenth, the fifteenth, thesixteenth, the seventeenth, the eighteenth, the nineteenth, thetwentieth and the twenty first embodiment of the first aspect, themethod further comprises protein precipitation, HPLC and MS/MS.

In a twenty third embodiment of the first aspect which is also anembodiment of the first, the second, the third, the fourth, the fifth,the sixth, the seventh, the eighth, the ninth, the tenth, the eleventh,the twelfth, the thirteenth, the fourteenth, the fifteenth, thesixteenth, the seventeenth, the eighteenth, the nineteenth, thetwentieth, the twenty first and the twenty second embodiment of thefirst aspect, the subject is a human.

In a twenty fourth embodiment of the first aspect which is also anembodiment of the first, the second, the third, the fourth, the fifth,the sixth, the seventh, the eighth, the ninth, the tenth, the eleventh,the twelfth, the thirteenth, the fourteenth, the fifteenth, thesixteenth, the seventeenth, the eighteenth, the nineteenth, thetwentieth, the twenty first, the twenty second and the twenty thirdembodiment of the first aspect, the step of detecting the biomarker in asample comprises subjecting the sample to a protein precipitation step,precipitating protein from the sample, wherein precipitating proteinfrom the sample provides a supernatant of the sample, subjecting thesupernatant of the sample to HPLC and MS/MS and determining the amountof the biomarker and/or the at least one additional biomarker thatis/are present in the supernatant of the sample.

The problem underlying the present invention is solved in a secondaspect which is also the first embodiment of the second aspect, by amethod for diagnosing Gaucher's disease in a subject comprising

i) adding an internal standard to a sample from the subject, wherein thesample form the subject is selected from the group comprising plasma,serum and blood;

ii) optionally mixing the sample containing the internal standard;

iii) subjecting the sample to a protein precipitation step, wherebyprotein from the sample is precipitated and a supernatant of the sampleis provided;

iv) optionally subjecting the supernatant of the sample to a firstseparation step which provides a supernatant, whereby preferably thefirst separation step is a step of centrifugation;

v) subjecting the supernatant of step c) or of step d), or a partthereof, to a second separation step, wherein the second separation stepcomprises injecting a part of the supernatant into an HPLC-MS/MS systemand using an HPLC column with a gradient form acidic water toacetonitrile/acetone; wherein the HPLC column is preferably an HPLCcolumn selected from the group comprising C8 and C18 HPLC column, andwherein the second separation step provides a separated sample;vi) subjecting the separated sample to MS/MS, wherein MS/MS compriseselectrospray ionization and Multiple Reacting Monitoring;wherein the method is preferably a method according to any one of thefirst, the second, the third, the fourth, the fifth, the sixth, theseventh, the eighth, the ninth, the tenth, the eleventh, the twelfth,the thirteenth, the fourteenth, the fifteenth, the sixteenth, theseventeenth, the eighteenth, the nineteenth, the twentieth, the twentyfirst, the twenty second, the twenty third and the twenty-fourthembodiment of the first aspect;and further comprising a step of

-   -   a) detecting a biomarker in a sample from the subject, wherein        the biomarker is free lyso-Gb1;        and optionally a step of    -   b) determining a level of the biomarker present in the sample.

In a second embodiment of the second aspect which is also an embodimentof the first embodiment of the second aspect, the internal standardcomprises D5-fluticasone propionate and/or lyso-Gb2.

In a third embodiment of the second aspect which is also an embodimentof the first and the second embodiment of the second aspect and of thefirst, the second, the third, the fourth, the fifth, the sixth, theseventh, the eighth, the ninth, the tenth, the eleventh, the twelfth,the thirteenth, the fourteenth, the fifteenth, the sixteenth, theseventeenth, the eighteenth, the nineteenth, the twentieth, the twentyfirst, the twenty second, the twenty third and the twenty fourthembodiment of the first aspect, the step of b) and/or the step of e)further comprises that the level of the biomarker in the sample from thesubject is compared to a cut-off level.

In a fourth embodiment of the second aspect which is also an embodimentof the first, the second and the third embodiment of the second aspectand of the first, the second, the third, the fourth, the fifth, thesixth, the seventh, the eighth, the ninth, the tenth, the eleventh, thetwelfth, the thirteenth, the fourteenth, the fifteenth, the sixteenth,the seventeenth, the eighteenth, the nineteenth, the twentieth, thetwenty first, the twenty second, the twenty third and the twenty fourthembodiment of the first aspect, preferably of the third embodiment ofthe second aspect, a level of the biomarker in the sample from thesubject which is higher than the cut-off level is indicative that thesubject is suffering from or is at risk for developing Gaucher'sdisease.

In a fifth embodiment of the second aspect which is also an embodimentof the fourth embodiment of the second aspect, a level of the biomarkerin the sample from the subject which is lower than the cut-off level isindicative that the subject is not suffering from or is not at risk fordeveloping Gaucher's disease.

In a sixth embodiment of the second aspect which is also an embodimentof the first, the second, the third, the fourth and the fifth embodimentof the second aspect and of the first, the second, the third, thefourth, the fifth, the sixth, the seventh, the eighth, the ninth, thetenth, the eleventh, the twelfth, the thirteenth, the fourteenth, thefifteenth, the sixteenth, the seventeenth, the eighteenth, thenineteenth, the twentieth, the twenty-first, the twenty-second, thetwenty-third and the twenty-fourth embodiment of the first aspect, thecut-off level is selected such that a sensitivity for diagnosingGaucher's disease in a subject is preferably from about 98.5% to 100%,more preferably 100% and that a specificity for diagnosing Gaucher'sdisease in a subject is preferably from 99.4% to 100%, more preferably100%.

In a seventh embodiment of the second aspect which is also an embodimentof the first, the second, the third, the fourth, the fifth and the sixthembodiment of the second aspect and of the first, the second, the third,the fourth, the fifth, the sixth, the seventh, the eighth, the ninth,the tenth, the eleventh, the twelfth, the thirteenth, the fourteenth,the fifteenth, the sixteenth, the seventeenth, the eighteenth, thenineteenth, the twentieth, the twenty-first, the twenty-second, thetwenty-third and the twenty-fourth embodiment of the first aspect, thestep of b) and/or the step of e) further comprises that a level of thebiomarker in said subject is compared to a level of the biomarkerdetected in a sample from a control.

In an eighth embodiment of the second aspect which is also an embodimentof the seventh embodiment of the second aspect, the control is a samplefrom a subject being positively tested for not having Gaucher's disease.

In a ninth embodiment of the second aspect which is also an embodimentof the first, the second, the third, the fourth, the fifth, the sixth,the seventh and the eighth embodiment of the second aspect and of thefirst, the second, the third, the fourth, the fifth, the sixth, theseventh, the eighth, the ninth, the tenth, the eleventh, the twelfth,the thirteenth, the fourteenth, the fifteenth, the sixteenth, theseventeenth, the eighteenth, the nineteenth, the twentieth, thetwenty-first, the twenty-second, the twenty-third and the twenty-fourthembodiment of the first aspect, a level of the biomarker in the samplefrom the subject which is higher than a level of the biomarker in thecontrol sample is indicative that the subject is suffering from and/oris at risk for developing Gaucher's disease.

In a tenth embodiment of the second aspect which is also an embodimentof the first, the second, the third, the fourth, the fifth, the sixth,the seventh, the eighth and the ninth embodiment of the second aspectand of the first, the second, the third, the fourth, the fifth, thesixth, the seventh, the eighth, the ninth, the tenth, the eleventh, thetwelfth, the thirteenth, the fourteenth, the fifteenth, the sixteenth,the seventeenth, the eighteenth, the nineteenth, the twentieth, thetwenty-first, the twenty-second, the twenty-third and the twenty-fourthembodiment of the first aspect, Gaucher's disease is selected from thegroup comprising the non-neuronopathic type I, the chronic neuronopathictype II and the acute neuronopathic type III.

In an eleventh embodiment of the second aspect which is also anembodiment of the first, the second, the third, the fourth, the fifth,the sixth, the seventh, the eighth, the ninth and the tenth embodimentof the second aspect and of the first, the second, the third, thefourth, the fifth, the sixth, the seventh, the eighth, the ninth, thetenth, the eleventh, the twelfth, the thirteenth, the fourteenth, thefifteenth, the sixteenth, the seventeenth, the eighteenth, thenineteenth, the twentieth, the twenty-first, the twenty-second, thetwenty-third and the twenty-fourth embodiment of the first aspect,preferably of the tenth embodiment of the second aspect, the sample fromthe subject is selected from the group consisting of blood, a bloodproduct, urine, saliva, cerebrospinal fluid, stool, tissue sample andlymph.

In a twelfth embodiment of the second aspect which is also an embodimentof the eleventh embodiment of the second aspect, the sample from thesubject is selected from the group consisting of blood and a bloodproduct.

In a thirteenth embodiment of the second aspect which is also anembodiment of the eleventh and the twelfth embodiment of the secondaspect, the blood product is selected from the group comprising serumand plasma.

In a fourteenth embodiment of the second aspect which is also anembodiment of the first, the second, the third, the fourth, the fifth,the sixth, the seventh, the eighth, the ninth, the tenth, the eleventh,the twelfth and the thirteenth embodiment of the second aspect and ofthe first, the second, the third, the fourth, the fifth, the sixth, theseventh, the eighth, the ninth, the tenth, the eleventh, the twelfth,the thirteenth, the fourteenth, the fifteenth, the sixteenth, theseventeenth, the eighteenth, the nineteenth, the twentieth, thetwenty-first, the twenty-second, the twenty-third and the twenty-fourthembodiment of the first aspect, preferably of the thirteenth embodimentof the second aspect, the method has a limit of detection of 0.2 ng/ml.

In a fifteenth embodiment of the second aspect which is also anembodiment of the first, the second, the third, the fourth, the fifth,the sixth, the seventh, the eighth, the ninth, the tenth, the eleventh,the twelfth, the thirteenth and the fourteenth embodiment of the secondaspect and of the first, the second, the third, the fourth, the fifth,the sixth, the seventh, the eighth, the ninth, the tenth, the eleventh,the twelfth, the thirteenth, the fourteenth, the fifteenth, thesixteenth, the seventeenth, the eighteenth, the nineteenth, thetwentieth, the twenty-first, the twenty-second, the twenty-third and thetwenty-fourth embodiment of the first aspect, preferably of any of theeleventh, the twelfth, the thirteenth, the fourteenth and the fifteenthembodiment of the second aspect, the cut-off level is 5.0 ng/ml.

In a sixteenth embodiment of the second aspect which is also anembodiment of the eleventh and the twelfth embodiment of the secondaspect, the blood is whole blood.

In an seventeenth embodiment of the second aspect which is also anembodiment of the seventeenth embodiment of the second aspect, the wholeblood is collected on a dry blood filter card.

In an eighteenth embodiment of the second aspect which is also anembodiment of the seventeenth and the eighteenth embodiment of thesecond aspect, the method has a limit of detection of 0.2 ng/ml.

In a nineteenth embodiment of the second aspect which is also anembodiment of the seventeenth, the eighteenth and the nineteenthembodiment of the second aspect, the cut-off level is 20.0 ng/ml.

The problem underlying the present invention is solved in a third aspectwhich is also the first embodiment of the third aspect, by a method fordetermining the course of Gaucher's disease in a subject comprising thestep of

-   -   a) determining at several points in time a level of a biomarker        present in a sample from the subject, wherein the biomarker is        free lyso-Gb1.

In a second embodiment of the third aspect which is also an embodimentof the first embodiment of the third aspect, the subject has beenpreviously treated or diagnosed for Gaucher's disease.

In a third embodiment of the third aspect which is also an embodiment ofthe first embodiment of the third aspect, the subject has not beenpreviously treated or wherein the subject has not been previouslydiagnosed for Gaucher's disease.

In a fourth embodiment of the third aspect which is also an embodimentof the first, the second and the third embodiment of the third aspect,the method further comprises a step of

-   -   b) applying, maintaining, reducing, elevating or not applying a        therapy based on the diagnosis of whether the subject is        suffering from or for being at risk for developing Gaucher's        disease.

In a fifth embodiment of the third aspect which is also an embodiment ofthe first, the second, the third and the fourth embodiment of the thirdaspect, the method further comprises a step of

-   -   c) detecting the biomarker in a sample from the subject after        applying, maintaining, reducing, elevating or not applying a        therapy in a step of b).

In a sixth embodiment of the third aspect which is also an embodiment ofthe first, the second, the third, the fourth and the fifth embodiment ofthe third aspect, the method further comprises a step of

-   -   d) determining a level of the biomarker in the sample from the        subject after applying, maintaining, reducing, elevating or not        applying a therapy in a step of b).

In a seventh embodiment of the third aspect which is also an embodimentof the first, the second, the third, the fourth, the fifth and the sixthembodiment of the third aspect, the method further comprises the stepsof

-   -   e) determining whether the level of the biomarker determined in        step a) is lower than the level of the biomarker determined in        step d);

In an eighth embodiment of the third aspect which is also an embodimentof the seventh embodiment of the third aspect, the method furthercomprises the step of

-   -   f) applying, maintaining, reducing, elevating or not applying a        therapy based on the step of e).

In a ninth embodiment of the third aspect which is also an embodiment ofthe first, the second, the third, the fourth, the fifth, the sixth, theseventh and the eighth embodiment of the third aspect, the methodfurther comprises detecting at least one additional biomarker in thesample from the subject.

In a tenth embodiment of the third aspect which is also an embodiment ofthe ninth embodiment of the third aspect, the method further comprisesdetermining the level of the at least one additional biomarker in thesample from the subject.

In an eleventh embodiment of the third aspect which is also anembodiment of the ninth and the tenth embodiment of the third aspect,the at least one additional biomarker is selected from the groupcomprising chitotriosidase and CCL18.

In a twelfth embodiment of the third aspect which is also an embodimentof the eleventh embodiment of the third aspect, the at least oneadditional biomarker is chitotriosidase.

In a thirteenth embodiment of the third aspect which is also anembodiment of the eleventh embodiment of the third aspect, the at leastone additional biomarker is CCL18.

In a fourteenth embodiment of the third aspect which is also anembodiment of the first, the second, the third, the fourth, the fifth,the sixth, the seventh, the eighth, the ninth, the tenth, the eleventh,the twelfth and the thirteenth embodiment of the third aspect, themethod further comprises detecting chitotriosidase and CCL18.

In a fifteenth embodiment of the third aspect which is also anembodiment of the first, the second, the third, the fourth, the fifth,the sixth, the seventh, the eighth, the ninth, the tenth, the eleventh,the twelfth, the thirteenth and the fourteenth embodiment of the thirdaspect, the biomarker and/or the at least one additional biomarker isdetected by means of immunoassay, mass spectrometric analysis, biochiparray, functional nucleic acids and/or a fluorescent derivative of freelyso-Gb1.

In a sixteenth embodiment of the third aspect which is also anembodiment of the fifteenth embodiment of the third aspect, thebiomarker is detected by means of mass spectrometric analysis.

In a seventeenth embodiment of the third aspect which is also anembodiment of the sixteenth embodiment of the third aspect, massspectrometric analysis is selected from the group consisting of SELDI,MALDI, MALDI-Q TOF, MS/MS, TOF-TOF and ESI-O-TOF.

In an eighteenth embodiment of the third aspect which is also anembodiment of the seventeenth embodiment of the third aspect, the massspectrometric analysis uses MS/MS.

In a nineteenth embodiment of the third aspect which is also anembodiment of the first, the second, the third, the fourth, the fifth,the sixth, the seventh, the eighth, the ninth, the tenth, the eleventh,the twelfth, the thirteenth, the fourteenth, the fifteenth, thesixteenth, the seventeenth and the eighteenth embodiment of the thirdaspect, the method further comprises protein precipitation and/or HPLC.

In a twentieth embodiment of the third aspect which is also anembodiment of the first, the second, the third, the fourth, the fifth,the sixth, the seventh, the eighth, the ninth, the tenth, the eleventh,the twelfth, the thirteenth, the fourteenth, the fifteenth, thesixteenth, the seventeenth, the eighteenth and the nineteenth embodimentof the third aspect, the method further comprises protein precipitation,HPLC and MS/MS.

In a twenty first embodiment of the third aspect which is also anembodiment of the first, the second, the third, the fourth, the fifth,the sixth, the seventh, the eighth, the ninth, the tenth, the eleventh,the twelfth, the thirteenth, the fourteenth, the fifteenth, thesixteenth, the seventeenth, the eighteenth, the nineteenth, thetwentieth and the twenty first embodiment of the third aspect, thesubject is a human.

In a twenty second embodiment of the third aspect which is also anembodiment of the first, the second, the third, the fourth, the fifth,the sixth, the seventh, the eighth, the ninth, the tenth, the eleventh,the twelfth, the thirteenth, the fourteenth, the fifteenth, thesixteenth, the seventeenth, the eighteenth, the nineteenth, thetwentieth, the twenty first and the twenty second embodiment of thethird aspect, the step of detecting the biomarker in the sample from thesubject comprises precipitating protein from the sample from thesubject, wherein precipitating protein from the sample provides asupernatant of the sample; subjecting a volume of the supernatant toHPLC and MS/MS and determining the amount of the biomarker and/or the atleast one additional biomarker that is/are present in the sample fromthe subject.

In a twenty third embodiment of the third aspect which is also anembodiment of the first, the second, the third, the fourth, the fifth,the sixth, the seventh, the eighth, the ninth, the tenth, the eleventh,the twelfth, the thirteenth, the fourteenth, the fifteenth, thesixteenth, the seventeenth, the eighteenth, the nineteenth, thetwentieth, the twenty first, the twenty second and the twenty thirdembodiment of the third aspect, Gaucher's disease is selected from thegroup comprising the non-neuronopathic type I, the chronic neuronopathictype II and the acute neuronopathic type III.

The problem underlying the present invention is solved in a fourthaspect which is also the first embodiment of the fourth aspect, by amethod for determining the effectiveness of at least one treatmentapplied to a subject being positively tested for suffering from or beingat risk for developing Gaucher's disease comprising the step of

-   -   a) determining at several points in time a level of a biomarker        present in a sample from the subject,        wherein the biomarker is free lyso-Gb1.

In a second embodiment of the fourth aspect which is also an embodimentof the first embodiment of the fourth aspect, the subject has beenpreviously treated or diagnosed for Gaucher's disease.

In a third embodiment of the fourth aspect which is also an embodimentof the first embodiment of the fourth aspect, the subject has not beenpreviously treated or wherein the subject has not been previouslydiagnosed for Gaucher's disease.

In a fourth embodiment of the fourth aspect which is also an embodimentof the first, the second and the third embodiment of the fourth aspect,the method further comprises a step of

-   -   b) applying, maintaining, reducing, elevating or not applying at        least one treatment applied to the subject based on the decrease        in the level of the biomarker.

In a fifth embodiment of the fourth aspect which is also an embodimentof the first, the second, the third and the fourth embodiment of thefourth aspect, the method further comprises a step of

-   -   c) detecting the biomarker in the sample from the subject,        wherein the sample has been taken prior to the beginning of the        treatment after applying, maintaining, reducing, elevating or        not applying at least one treatment in a step of b).

In a sixth embodiment of the fourth aspect which is also an embodimentof the first, the second, the third, the fourth and the fifth embodimentof the fourth aspect, the treatment is selected from the groupcomprising enzyme replacement therapy, substrate reduction therapy,chaperone therapy, gene therapy, stem cell transplantation of DNA/RNAskipping.

In a seventh embodiment of the fourth aspect which is also an embodimentof the first, the second, the third, the fourth, the fifth and the sixthembodiment of the fourth aspect, the method further comprises the stepsof

-   -   d) determining whether the level of the biomarker determined in        step a) is lower than the level of the biomarker determined in        step c).

In an eighth embodiment of the fourth aspect which is also an embodimentof the seventh embodiment of the fourth aspect, the method furthercomprises the steps of

-   -   e) applying, maintaining, reducing, elevating or not applying at        least one treatment applied to the subject based on the step of        d).

In a ninth embodiment of the fourth aspect which is also an embodimentof the first, the second, the third, the fourth, the fifth, the sixth,the seventh and the eighth embodiment of the fourth aspect, the methodfurther comprises detecting at least one additional biomarker in thesample from the subject.

In a tenth embodiment of the fourth aspect which is also an embodimentof the ninth embodiment of the fourth aspect, the method furthercomprises determining the level of the at least one additional biomarkerin the sample from the subject.

In an eleventh embodiment of the fourth aspect which is also anembodiment of the ninth and the tenth embodiment of the fourth aspect,the at least one additional biomarker is selected from the groupcomprising chitotriosidase and CCL18.

In a twelfth embodiment of the fourth aspect which is also an embodimentof the eleventh embodiment of the fourth aspect, the at least oneadditional biomarker is chitotriosidase.

In a thirteenth embodiment of the fourth aspect which is also anembodiment of the eleventh h embodiment of the fourth aspect, the atleast one additional biomarker is CCL18.

In a fourteenth embodiment of the fourth aspect which is also anembodiment of the first, the second, the third, the fourth, the fifth,the sixth, the seventh, the eighth, the ninth, the tenth, the eleventh,the twelfth and the thirteenth embodiment of the fourth aspect, themethod further comprises detecting chitotriosidase and CCL18.

In a fifteenth embodiment of the fourth aspect which is also anembodiment of the first, the second, the third, the fourth, the fifth,the sixth, the seventh, the eighth, the ninth, the tenth, the eleventh,the twelfth, the thirteenth and the fourteenth embodiment of the fourthaspect, any/the biomarker is detected by means of immunoassay, massspectrometric analysis, biochip array, functional nucleic acids and/or afluorescent derivative of free lyso-Gb1.

In a sixteenth embodiment of the fourth aspect which is also anembodiment of the fifteenth embodiment of the fourth aspect, thebiomarker is detected by means of mass spectrometric analysis.

In a seventeenth embodiment of the fourth aspect which is also anembodiment of the sixteenth embodiment of the fourth aspect, massspectrometric analysis is selected from the group consisting of SELDI,MALDI, MALDI-Q TOF, MS/MS, TOF-TOF and ESI-O-TOF.

In an eighteenth embodiment of the fourth aspect which is also anembodiment of the seventeenth embodiment of the fourth aspect, the massspectrometric analysis uses MS/MS.

In a nineteenth embodiment of the fourth aspect which is also anembodiment of the first, the second, the third, the fourth, the fifth,the sixth, the seventh, the eighth, the ninth, the tenth, the eleventh,the twelfth, the thirteenth, the fourteenth, the fifteenth, thesixteenth, the seventeenth and the eighteenth embodiment of the fourthaspect, the method further comprises protein precipitation and/or HPLC.

In a twentieth embodiment of the fourth aspect which is also anembodiment of the first, the second, the third, the fourth, the fifth,the sixth, the seventh, the eighth, the ninth, the tenth, the eleventh,the twelfth, the thirteenth, the fourteenth, the fifteenth, thesixteenth, the seventeenth, the eighteenth and the nineteenth embodimentof the fourth aspect, the method further comprises proteinprecipitation, HPLC and MS/MS.

In a twenty-first embodiment of the fourth aspect which is also anembodiment of the first, the second, the third, the fourth, the fifth,the sixth, the seventh, the eighth, the ninth, the tenth, the eleventh,the twelfth, the thirteenth, the fourteenth, the fifteenth, thesixteenth, the seventeenth, the eighteenth, the nineteenth and thetwentieth embodiment of the fourth aspect, the subject is a human.

In a twenty-second embodiment of the fourth aspect which is also anembodiment of the first, the second, the third, the fourth, the fifth,the sixth, the seventh, the eighth, the ninth, the tenth, the eleventh,the twelfth, the thirteenth, the fourteenth, the fifteenth, thesixteenth, the seventeenth, the eighteenth, the nineteenth, thetwentieth and the twenty-first embodiment of the fourth aspect, the stepof detecting the biomarker in the sample from the subject comprisesprecipitating protein from the sample from the subject, whereinprecipitating protein from the sample provides a supernatant of thesample; subjecting a volume of the supernatant to HPLC and MS/MS anddetermining the amount of the biomarker and/or the at least oneadditional biomarker that is/are present in the sample from the subject.

In a twenty-third embodiment of the fourth aspect which is also anembodiment of the first, the second, the third, the fourth, the fifth,the sixth, the seventh, the eighth, the ninth, the tenth, the eleventh,the twelfth, the thirteenth, the fourteenth, the fifteenth, thesixteenth, the seventeenth, the eighteenth, the nineteenth, thetwentieth, the twenty-first and the twenty-second embodiment of thefourth aspect, Gaucher's disease is selected from the group comprisingthe non-neuronopathic type I, the chronic neuronopathic type II and theacute neuronopathic type III.

The problem underlying the present invention is solved in a fifth aspectwhich is also the first embodiment of the fifth aspect, by a method ofdetermining the effectiveness of a compound for the treatment ofGaucher's disease comprising the steps of:

-   -   a) determining a level of a biomarker in a subject having        Gaucher's disease;    -   b) administering to said subject said compound;    -   c) determining again the level of the biomarker in said subject;    -   d) determining whether the level of the biomarker determined in        step a) is lower than the level of the biomarker determined in        step c),        wherein a level of the biomarker determined in step c) which is        lower than the level of the biomarker determined in step a)        indicates the effectiveness of said compound, and wherein the        biomarker is free lyso-Gb1.

In a second embodiment of the fifth aspect which is also an embodimentof the first embodiment of the fifth aspect, the method furthercomprises determining a level of the biomarker in a control.

In a third embodiment of the fifth aspect which is also an embodiment ofthe first and the second embodiment of the fifth aspect, Gaucher'sdisease is selected from the group comprising the non-neuronopathic typeI, the chronic neuronopathic type II and the acute neuronopathic typeIII.

The problem underlying the present invention is solved in a sixth aspectwhich is also the first embodiment of the sixth aspect, by the use ofmass spectrometry for the detection of a biomarker, wherein thebiomarker is free lyso-Gb1.

In a second embodiment of the sixth aspect which is also an embodimentof the first embodiment of the sixth aspect, the detection comprises theuse of HPLC.

In a third embodiment of the sixth aspect which is also an embodiment ofthe first and the second embodiment of the sixth aspect, the detectioncomprises MS/MS.

The problem underlying the present invention is solved in a seventhaspect which is also the first embodiment of the seventh aspect, by theuse of a biomarker for Gaucher's disease, preferably in a methodaccording to any one of the first, the second, the third, the fourth,the fifth, the sixth, the seventh, the eighth, the ninth, the tenth, theeleventh, the twelfth, the thirteenth, the fourteenth, the fifteenth,the sixteenth, the seventeenth, the eighteenth, the nineteenth, thetwentieth, the twenty-first, the twenty-second, the twenty-third and thetwenty-fourth embodiment of the first aspect, of the first, the second,the third, the fourth, the fifth, the sixth, the seventh, the eighth,the ninth, the tenth, the eleventh, the twelfth, the thirteenth, thefourteenth, the fifteenth, the sixteenth, the seventeenth, theeighteenth and the nineteenth embodiment of the second aspect, of thefirst, the second, the third, the fourth, the fifth, the sixth, theseventh, the eighth, the ninth, the tenth, the eleventh, the twelfth,the thirteenth, the fourteenth, the fifteenth, the sixteenth, theseventeenth, the eighteenth, the nineteenth, the twentieth, thetwenty-first, the twenty-second and the twenty-third embodiment of thethird aspect, of the first, the second, the third, the fourth, thefifth, the sixth, the seventh, the eighth, the ninth, the tenth, theeleventh, the twelfth, the thirteenth, the fourteenth, the fifteenth,the sixteenth, the seventeenth, the eighteenth, the nineteenth, thetwentieth, the twenty-first, the twenty-second and the twenty-thirdembodiment of the fourth aspect and of the first, the second and thethird embodiment of the fifth aspect, wherein the biomarker is freelyso-Gb1.

In a second embodiment of the seventh aspect which is also an embodimentof the first embodiment of the seventh aspect, Gaucher's disease isselected from the group comprising the non-neuronopathic type I, thechronic neuronopathic type II and the acute neuronopathic type III.

The problem underlying the present invention is solved in a eighthaspect which is also the first embodiment of the eight aspect, by a kitfor determining the presence of a biomarker in a sample from a subject,wherein the kit comprises

-   -   a) an interaction partner of the biomarker;    -   b) optionally a solid support comprising at least one capture        reagent attached thereto, wherein the capture reagent binds the        biomarker; and    -   c) instructions for using the solid support to detect the        biomarker,        wherein the biomarker is free lyso-Gb1.

In a second embodiment of the eighth aspect which is also an embodimentof the first embodiment of the eighth aspect, the kit is for

-   -   a) diagnosing Gaucher's disease;    -   b) determining the course of Gaucher's disease in a subject;        and/or    -   c) determining the effectiveness of at least one treatment        applied to a subject,        wherein a method applied in a), b) and/or c) is preferably a        method according to any one of the first, the second, the third,        the fourth, the fifth, the sixth, the seventh, the eighth, the        ninth, the tenth, the eleventh, the twelfth, the thirteenth, the        fourteenth, the fifteenth, the sixteenth, the seventeenth, the        eighteenth, the nineteenth, the twentieth, the twenty-first, the        twenty-second, the twenty-third and the twenty-fourth embodiment        of the first aspect, of the first, the second, the third, the        fourth, the fifth, the sixth, the seventh, the eighth, the        ninth, the tenth, the eleventh, the twelfth, the thirteenth, the        fourteenth, the fifteenth, the sixteenth, the seventeenth, the        eighteenth and the nineteenth embodiment of the second aspect,        of the first, the second, the third, the fourth, the fifth, the        sixth, the seventh, the eighth, the ninth, the tenth, the        eleventh, the twelfth, the thirteenth, the fourteenth, the        fifteenth, the sixteenth, the seventeenth, the eighteenth, the        nineteenth, the twentieth, the twenty-first, the twenty-second        and the twenty-third embodiment of the third aspect, of the        first, the second, the third, the fourth, the fifth, the sixth,        the seventh, the eighth, the ninth, the tenth, the eleventh, the        twelfth, the thirteenth, the fourteenth, the fifteenth, the        sixteenth, the seventeenth, the eighteenth, the nineteenth, the        twentieth, the twenty-first, the twenty-second and the        twenty-third embodiment of the fourth aspect and of the first,        the second and the third embodiment of the fifth aspect.

In a third embodiment of the eighth aspect which is also an embodimentof the first and the second embodiment of the eighth aspect, Gaucher'sdisease is selected from the group comprising the non-neuronopathic typeI, the chronic neuronopathic type II and the acute neuronopathic typeIII.

The problem underlying the present invention is solved in a ninth aspectwhich is also the first embodiment of the ninth aspect, by a softwareproduct comprising

-   -   a) code that accesses data attributed to a sample, the data        comprising detection of at least one biomarker in the sample,        the biomarker selected from the group comprising free lyso-Gb1,        Chitotriosidase and CCL18; and    -   b) code that executes a classification algorithm that classifies        Gaucher's disease status of the sample as a function of the        detection.

In a second embodiment of the ninth aspect which is also an embodimentof the first embodiment of the ninth aspect, Gaucher's disease isselected from the group comprising the non-neuronopathic type I, thechronic neuronopathic type II and the acute neuronopathic type III.

The present inventors have surprisingly found that free lyso-Gb1constitutes a biomarker which allows for a method for diagnosingGaucher's disease in a subject, more specifically diagnosing Gaucher'sdisease in a subject with high specificity and sensitivity using saidfree lyso-Gb1 as the biomarker.

The present inventors have also surprisingly found that free lyso-Gb1,which can be detected by the methods of the present invention, iscirculating in the blood of a subject in a concentration ofapproximately 1/1000 of total Gb1. Moreover, the present inventors havesurprisingly found that, unlike total Gb1, free lyso-Gb1 which ispresent in the blood of a subject is useful in a method for diagnosingGaucher's disease in a subject comprising a step of detecting abiomarker in a sample from the subject, wherein the biomarker is freelyso-Gb1. The present inventors have also surprisingly found that thelevel of free lyso-Gb1 determined in the sample from a subject by themethods of the present invention allows for diagnosing Gaucher's diseasewith high sensitivity and high specificity.

In so far the present invention turns away from the teaching of thestate of the art in that the method of the present invention comprisesdetermining the level of a lyso-compound using said lyso-compound as abiomarker for diagnosis of a sphingolipidoses. More specifically, thepresent inventors have surprisingly found that determining the level offree lyso-Gb1 in a sample from a subject allows for diagnosing Gaucher'sdisease with high sensitivity and high specificity.

It is also the merit of the present inventors of having recognized thata fraction of total Gb1 which is accumulated in Gaucher's disease, ispresent as a molecule in a free lyso form thereof, i.e. free lyso-Gb1,and is circulating in the blood of a subject in said free lyso formbesides Gb1.

The term “lysosomal storage disorder”, also referred to as “lysosomalstorage disease” or “LSD”, as used herein, preferably refers to geneticdiseases and metabolic disorders that result from defects in lysosomalfunction. Lysosomal storage disorders are caused by lysosomaldysfunction usually as a consequence of deficiency of a single enzymerequired for the metabolism of lipids, glycoproteins or so-calledmucopolysaccharides. Like other genetic diseases, individuals inheritlysosomal storage diseases from their parents. Although each disorderresults from different gene mutations that translate into a deficiencyin enzyme activity, they all share a common biochemicalcharacteristic—all lysosomal disorders originate from an abnormalaccumulation of substances inside the lysosome.

The term “Gaucher's disease” as used herein, preferably refers to alysosomal storage disease (LSD), more specifically a sphingolipidosesthat is characterized by the deposition of glucocerebroside in cells ofthe macrophage-monocyte system. Gaucher's disease is the most common ofthe lysosomal storage diseases (James, William D.; Berger, Timothy G.;et al. (2006). Andrews' Diseases of the Skin: clinical Dermatology.Saunders Elsevier. ISBN 0-7216-2921-0). It is caused by a hereditarydeficiency of the enzyme glucocerebrosidase. Said deficiency resultsfrom recessive mutation(s) in the gene coding for glucocerebrosidase, aspecific lysosomal hydrolase (also known as beta-glucosidase, EC3.2.1.45, PDB 1OGS) located on chromosome 1 (1q21) and affects bothmales and females. Different mutations in the beta-glucosidase determinethe remaining activity of the enzyme, and, to a large extent, thephenotype.

Glucocerebrosidase is also referred to herein as β-glucocerebrosidase,beta-glucosidase, acid beta-glucosidase, glucosylceramidase orD-glucosyl-N-acylsphingosine glucohydrolase. The enzyme is a 55.6 KD,497 amino acids long protein having glucosylceramidase activity, i.e.the enzyme catalyses the breakdown of a fatty substance calledglucocerebroside by cleavage, i.e. hydrolysis, of a beta-glucosidiclinkage of glucocerebroside, which is an intermediate in glycolipidmetabolism. Glucocerebroside, also referred to herein asglucosylceramide or Gb1, is a cell membrane constituent of red and whiteblood cells. When the enzyme is defective, the substance accumulates,particularly in cells of the mononuclear cell lineage. This is becausemacrophages that clear these cells are unable to eliminate the wasteproduct, which accumulates in fibrils, and turn into so called Gauchercells, which appear on light microscopy to resemble crumpled-up paper.Fatty material can accumulate in the spleen, liver, kidneys, lungs,brain and bone marrow.

Gaucher's disease has three common clinical subtypes.

-   -   Non-neuronopathic type I, also referred to herein as type I, is        the most common form of the disease, occurring in approximately        1 in 50,000 live births. It occurs most often among persons of        Ashkenazi Jewish heritage. Symptoms may begin early in life or        in adulthood and include enlarged liver and grossly enlarged        spleen (together hepatosplenomegaly); the spleen can rupture and        cause additional complications. Skeletal weakness and bone        disease may be extensive. Spleen enlargement and bone marrow        replacement cause anemia, thrombocytopenia and leukopenia. The        brain is not affected pathologically, but there may be lung and,        rarely, kidney impairment. Diseased subjects in this group        usually bruise easily (due to low levels of platelets) and        experience fatigue due to low numbers of red blood cells.        Depending on disease onset and severity, type I patients may        live well into adulthood. Many diseased subjects have a mild        form of the disease or may not show any symptoms.    -   Chronic neuronopathic type II, also referred to herein as type        II, can begin at any time in childhood or even in adulthood, and        occurs in approximately 1 in 100,000 live births. It is        characterized by slowly progressive but milder neurologic        symptoms compared to the acute or type III version. Major        symptoms include an enlarged spleen and/or liver, seizures, poor        coordination, skeletal irregularities, eye movement disorders,        blood disorders including anemia and respiratory problems.        Patients often live into their early teen years and adulthood.    -   Acute neuronopathic type III, also referred to herein as type        III, typically begins within 6 months of birth and has an        incidence rate of approximately 1 in 100,000 live births.        Symptoms include an enlarged liver and spleen, extensive and        progressive brain damage, eye movement disorders, spasticity,        seizures, limb rigidity, and a poor ability to suck and swallow.        Affected children usually die by age 2.

These subtypes have come under some criticism for not taking account ofthe full spectrum of observable symptoms. There are also compoundheterozygous variations which considerably increase the complexity ofpredicting disease course.

In type II and III of Gaucher's disease, glucocerebroside accumulatesthe brain due to the turnover of complex lipids during brain developmentand the formation of the myelin sheath of nerves.

Symptoms may include enlarged spleen and liver, liver malfunction,skeletal disorders and bone lesions that may be painful, severeneurologic complications, swelling of lymph nodes and (occasionally)adjacent joints, distended abdomen, a brownish tint to the skin, anemia,low blood platelets and yellow fatty deposits on the white of the eye(sclera). Persons affected most seriously may also be more susceptibleto infection.

Therapy: Enzyme replacement treatment also referred to herein as ERT, isthe therapy of choice. However, successful bone marrow transplantationmight cure the non-neurological manifestations of the disease, becauseit introduces a monocyte population with active beta-glucosidase. It isimportant to mention that this procedure carries significant risk and israrely performed in Gaucher's disease patients. Surgery to remove thespleen (splenectomy) may be very rarely required if the patient ismassively anemic or when the enlarged organ affects the patient'scomfort. Blood transfusion may benefit some anemic patients. Otherpatients may require joint replacement surgery to improve mobility andquality of life. Other treatment options include antibiotics forinfections, antiepileptics for seizures, bisphosphonates for bonelesions, and liver transplants.

ERT is based on chronic intravenous administration of a recombinantglucocerebrosidase (imiglucerase, Genzyme; velaglucerase, Shire;taliglucerase, Protalix) (G. A. Grabowski et al., Enzyme therapy in typeI Gaucher's disease: comparative efficacy of mannose-terminatedglucocerebrosidase from natural and recombinant sources, Ann. Intern.Med. 122 (1995) 33-39.). For type I and most type III patients, ERT withintravenous recombinant glucocerebrosidase (such as, e.g., imiglucerase)can significantly reduce liver and spleen size, reduce skeletalabnormalities, and reverse other manifestations.

More recently substrate reduction therapy also referred to herein asSRT, has been developed as an alternative treatment for Gaucher'sdisease (F. M. Platt et al. N-butyl-deoxynojirirnycin is a novelinhibitor of glycosphingolipid biosynthesis, J. Biol. Chem. 269 (1994)8362-8365.). Partial inhibition of glycosphingolipid synthesis withN-butyl-deoxynojirimycin (miglustat, Actelion) is employed in an effortto balance the reduced catabolic capacity in Gaucher's disease patients.SRT may prove to be effective in stopping type II, as it can crossthrough the blood barrier into the brain. There is currently noeffective treatment for the severe brain damage that may occur inpatients with types II and III Gaucher's disease.

Both ERT and SRT generally result in marked clinical improvements suchas reduction in hepatosplenomegaly, corrections in hematologicalabnormalities, stabilization or improvement in skeletal deterioration.

Glucocerebroside, also referred to herein as glucosylceramide or Gb1,means any cerebroside in which the monosaccharide head group is glucose.

It will be understood by a person skilled in the art that the term“lyso-Gb1” as used herein, preferably in connection with the variousmethods, preferably means that the molecule is present in its free aminoform. More precisely, lyso-Gb1 as used herein, preferably differs fromGb1 in that no fatty acid moiety is linked to the primary amino group ofthe sphingosine moiety of the molecule. Furthermore, lyso-Gb1 is alsoreferred to herein as glucosylsphingosine or lyso-glucocerebroside andhas the formula:

It will be understood by a person skilled in the art that the term “freelyso-Gb1” as used herein preferably refers to lyso-Gb1 which is as suchpresent in a sample from the subject, such as blood, and, preferably,not the result of a manipulation of the sample of said subject. Suchmanipulation of a sample can be the one described by Groener et al.(Groener et al. Plasma glucosylceramide and ceramide in type 1 Gaucherdisease patients: Correlations with disease severity and response totherapeutic intervention. Biochimica et Biophysica Acta 1781(2908)72 78,2007). In accordance therewith, free lyso-Gb1 which is present as suchin the blood of a subject from whom the sample is taken, is moreparticularly not a lyso-Gb1 which is generated by chemical, biochemicalor physical treatment of the sample contained in the blood and sample,respectively, preferably outside of the body of the patient. It will bealso understood by a person skilled in the art that free lyso-Gb1 asused herein, preferably is present in addition to Gb1 and is a compoundproduced by the subject's metabolic activities. Accordingly, Gb1, whichis the molecule that is accumulated in connection with Gaucher's diseaseis present in the sample from the subject has compared to the moleculein a free lyso form, i.e. free-lyso-Gb1, present in the blood of thesubject at least one fatty acid moiety linked to the primary amino groupof the sphingosine moiety of lyso-Gb1.

The term “sample” as preferably used herein means a limited quantity ofa subject's material, wherein said subject's material is part of or hasbeen taken from a subject and/or a subject's body and wherein saidmaterial is selected from the group comprising body fluids such asblood, a blood product, urine, saliva, cerebrospinal fluid and lymph, aswell as stool or any kind of tissue and or cell material being part of asubject and/or a subject's body. It will be acknowledged by a personskilled in the art, that the presence of and/or a level of a biomarkerof the invention in said sample is intended to be similar to andrepresent the presence and/or the level of the biomarker in a largeramount of that subject's material. More precisely and as anillustrative, non-limiting example, a level of a biomarker of theinvention determined in a sample of some ml of blood from a subject alsorepresents a level of said biomarker in the blood of the subject's body.Furthermore, in an embodiment of the method of the invention fordiagnosing Gaucher's disease in a subject, a sample from the subjectcomprises said subject's material in a form, for example processed,fixed and/or preserved such that said sample is suitable for use in themethod of the invention, whereby such processing, fixing and/orpreserving preferably does not generate lyso-Gb1. The subject's materialin the sample may thus be diluted, for example with a solvent suitablefor the method of the invention such as methanol and/or water, may bedried, for example on a filter card, may be resolved after having beendried such, for example with a solvent suitable for the method of theinvention such as methanol and/or water, or a substance may be added,wherein said substance prevents blood from coagulation such as forexample EDTA or heparin. It will be further understood by a personskilled in the art that the method of the invention comprises that saidsubject's material is separated into single components of said subject'smaterial and/or single components of said subject's material areextracted from said subject's material, for example blood is separatedinto plasma or serum and cellular blood components or protein isprecipitated from the sample. It will be immediately understood thatafter such processing, fixing and/or preserving the sample is subjectedto the methods of the invention for detecting and/or determining thelevel of a biomarker contained in said sample whereby such processing,fixing and/or preserving preferably does not generate lyso-Gb1.

In an embodiment of the method of the present invention wherein wholeblood is collected on a dry blood filter card preferably approximately 3μl of full blood are collected on a spot of said dry blood filter cardhaving a diameter of 3 mm A person skilled in the art will acknowledgethat the exact volume thus collected may vary depending on thehematocrit of the specific patient.

The levels of glucosylceramide and its precursor ceramide were used inthe prior art to correlate their presence in plasma with the severity ofGaucher's disease type I and the response to the application of therapy(Groener et al., Plasma glucosylceramide and ceramide in type 1Gaucher's disease patients: Correlations with disease severity andresponse to therapeutic intervention. Biochimica et Biophysica Acta1781(2908)72˜78, 2007). Thereby, the level of Gb1 was found to bedifferent although ceramide levels were not significantly different inthe plasma of treated and untreated Gaucher's disease type I patients.

In the study reported by Groener et al. (Groener et al., supra) theratio of Gb1/ceramide was used to discriminate between Gaucher's diseasepatients and healthy patients. Gb1 and ceramide were measured with highperformance liquid chromatography (HPLC) essentially as described inGroener et al. (J. E. M. Groener et al., HPLC for simultaneousquantification of total ceramide, glucosylceramide, and ceramidetrihexoside concentrations in plasma, Clin. Chem. 53 (2007) 742-747). Inconnection therewith it is important to understand that Gb1 present inthe plasma mainly consists of a sugar moiety and a ceramide moiety. Theceramide moiety comprising a sphingosine and a fatty acid moiety.According to the method of the prior art lipids are extracted andceramide and glucosylceramide are deacylated by alkaline hydrolysis thusforming the lyso form, i.e. lyso-Gb1 (T. Taketomi et al., Rapid methodof preparation of lysoglycosphingolipids and their confirmation bydelayed extraction matrix-assisted laser desporption ionizationtime-of-flight mass spectrometry, J. Biochem. (Tokyo) 120 (1996)573-579). Subsequently, the thus produced lyso-Gb1 is labeled with afluorescence dye by derivatization with O-phthaldialdehyde (OPA) at theprimary amine group. Afterwards the derivatized sphingoid bases wereseparated by reverse phase HPLC and detected with a fluorescencedetector. Thus said method of the prior art is able to detect total Gb1consisting of free lyso-Gb1 and Gb1 and is not able to distinguish alevel of free lyso-Gb1 from a level of Gb1 in a sample from a subject.The level of said total Gb1 after cleavage of the various fatty acidmoieties from the NH2 group of the Gb1 is usually in a range of from 5to 30 μg per mL plasma or serum. From this it is evident that in themethod of Groener et al. (Groener et al., supra) the total-Gb1 which canbe prepared and obtained, respectively, from a sample, preferably ablood sample, from a subject is used as a biomarker rather than the freelyso-Gb1 contained in the blood and accordingly also in the samplewithout performing a cleavage of the fatty acid moiety/moieties,preferably a cleavage performed by an operator handling the sample.Insofar, the present invention is related to the detection of freelyso-Gb1 rather than total-Gb1 as taught in the prior art. It is anembodiment of the methods of the present invention comprising detectingand/or determining the level of free lyso-Gb1 in a sample from a subjectthat free lyso-Gb1 and/or the level of free lyso-Gb1 is determinedseparate from and/or apart from Gb1 or a level of Gb1 which may bepresent in the blood of a subject. In a further embodiment Gb1 and/or alevel of Gb1 is detected/determined in addition to the detection ofand/or the determining of a level of free lyso-Gb1.

Importantly, each primary amine circulating in the plasma and beingsufficiently lipophilic to be extracted concomitantly with Gb1 using anorganic solvent according to said method of the art is labeledaccordingly and thus is able to disturb the detection of cleavedlyso-Gb1.

Although total Gb1 measured as lyso-Gb1 in said study of the prior artwas increased in plasma of said patients, said increase in total Gb1 wasnot prominent and thus the specificity and the sensitivity of the methodwere low showing that Gb1 is not suitable as a biomarker for Gaucher'sdisease.

In connection therewith it is important to note that to the knowledge ofthe inventors the data described in the Examples herein in connectionwith the present invention represent the first systematic analysis ofspecificity and sensitivity with regard to a direct comparison ofbiomarkers for Gaucher's disease of the prior art, i.e. chitotriosidaseand CCL18, and of free lyso-Gb1.

Providing a sensitivity and/or specificity of ≥99.0% free lyso-Gb1 asdetermined by the methods of the present invention is a biomarkersuitable of clinical application in connection with Gaucher's disease.Insofar, the biomarker of the present invention and uses thereof clearlyexceed the performance of biomarkers known the prior art, morespecifically, the one of chitotriosidase and CCL18. It will beimmediately understood that also the method applied by Groener et al.(Groener et al., supra) is prejudicial compared to the methods of thepresent invention in that the specificity and sensitivity of said methodof the prior art is lower and diagnosing of Gaucher's disease based onsuch method of the prior art using total Gb1 rather than free lyso-Gb1is not suitable for reliable clinical application thereof, i.e. themethod has no sensitivity and specificity sufficient to diagnoseGaucher's disease by a reliable statistically secured prediction.

Chitotriosidase: It has been found previously that Gaucher cells secretechitotriosidase and that chitotriosidase in plasma of symptomaticpatients with Gaucher's disease is elevated on average severalhundred-fold (Hollak et al. Marked elevation of plasma chitotriosidaseactivity. A novel hallmark of Gaucher disease. J Clin Invest. 1994; 93:1288-1292). Therefore, plasma chitotriosidase is used therefore as asurrogate marker for Gaucher's disease manifestations and is used fordiagnosis, early determination of onset of disease, and monitoring oftherapeutic efficacy (Hollak et al. Marked elevation of plasmachitotriosidase activity. A novel hallmark of Gaucher disease. J ClinInvest. 1994; 93: 1288-1292; Mistry et al. A practical approach todiagnosis and management of Gaucher's disease. Baillieres Clin Haematol.1997; 10: 817-838; Cox et al. Novel oral treatment of Gaucher's diseasewith N-butyldeoxynojirimycin (OGT 918) to decrease substratebiosynthesis. Lancet. 2000; 355: 1481-1485; Hollak et al. Clinicallyrelevant therapeutic endpoints in type I Gaucher disease. J InheritMetab Dis. 2001; 24: 97-105).

Nevertheless, plasma chitotriosidase levels do not reflect oneparticular clinical symptom, but rather are a reflection of the totalbody burden of Gaucher cells (Aerts et al. Plasma and metabolicabnormalities in Gaucher's disease. Baillieres Clin Haematol. 1997; 10:691-709); furthermore it is not reflecting the burden of the diseasedriven by the bone pathology and the brain damage. The level ofchitotriosidase is not directly linked to the pathophysiology ofGaucher's disease. Additionally, after treatment the level ofchitotriosidase changes extremely slowly making chitotriosidaseunsuitable for assessing quickly the efficacy of treatment to which thepatient is or has been subjected as well as a relapse of the diseaseindependent form the cause of the disease.

Furthermore, the use of plasma chitotriosidase as a Gaucher cell markeris hampered by the fact that patients, including those with Gaucher'sdisease, may be deficient in chitotriosidase activity due to a 24-basepair (bp) duplication in the chitotriosidase gene. Obviously theseindividuals cannot be monitored by the measurement of plasmachitotriosidase activity (Hollak et al. Marked elevation of plasmachitotriosidase activity. A novel hallmark of Gaucher disease. J ClinInvest. 1994; 93: 1288-1292; Boot et al. The human chitotriosidase gene.Nature of inherited enzyme deficiency. J Biol Chem. 1998; 273:25680-25685). The frequency of the 24-bp duplication in thechitotriosidase gene depends on the ethnicity and can raise up to nearly35% (Prof. Guiliani, Brasil, unpublished data).

CCL18: The glucosylceramide-laden macrophages or Gaucher cells are themain source of CCL18. The level of CCL18 in the plasma of patientshaving Gaucher's disease is significantly increased (Boot, R. G. et al.2004. Marked elevation of the chemokine CCL18/PARC in Gaucher disease: anovel surrogate marker for assessing therapeutic intervention. Blood103:33-39.). Therefore there were attempts to use the level of CCL18 inthe plasma as a surrogate marker for monitoring the success of a therapyapplied. Nevertheless, elevated levels of CCL18 were also found to beassociated with a variety of disease, such as different types of cancerand inflammation of joints, lungs and skin. For example, ascites ofpatients having ovarian carcinoma contains a significantly elevatedlevel of CCL18 compared to patients without ovarian carcinoma(Budd-Chiari syndrome) (Schutyser, E. et al. 2002. Identification ofbiologically active chemokine isoforms from ascitic fluid and elevatedlevels of CCL18/pulmonary and activation-regulated chemokine in ovariancarcinoma. J Biol. Chem. 277:24584-24593.). CCL18 plays a role in tumorsuppression since it attracts and activates specific immune cells.Furthermore children having acute lymphocytic leukemia are found toexhibit elevated levels of CCL18, whereas children having acute myeloidleukemia do not show elevated serum levels of CCL18 (Struyf, S et al.2003. ARC/CCL18 is a plasma CC chemokine with increased levels inchildhood acute lymphoblastic leukemia. Am J Pathol. 163: 2065-2075.).Again plasma CCL18 levels do not reflect one particular clinicalsymptom, but rather are a reflection of the total body burden of Gauchercells. As can be seen from the above CCL18 exhibits an extremely lowspecificity for the diagnosis of Gaucher's disease and is thus mainlyapplied as a “auxiliary” surrogate marker for patients deficient inchitotriosidase activity.

In connection with the use of chitotriosidase and CCL18 it is to benoted that chitotriosidase fails testing positive in 10-30% of allpatients, i.e. patients are tested negative although suffering fromGaucher's disease and thus also the application of a therapy will berenounced. Furthermore, in these cases the marker cannot further be usedas a follow-up marker for monitoring, e.g., ERT. If it is suspected thatthe patient is affected by the defect of chitotriosidase, CCL18 is usedas a biomarker for diagnosing Gaucher's disease, whereby a method makinguse of CCL18 as a biomarker exhibits relatively low specificity andsensitivity, i.e. diagnoses false positive or false negative in about25% of all patients.

The term “Gaucher's disease status” as used herein, preferably refers tothe status of the disease in the subject. Examples of types of Gaucher'sdisease statuses include, but are not limited to, the subject's risk ofsuffering or developing Gaucher's disease, the stage of the disease in asubject and the effectiveness of treatment of the disease. Otherstatuses and degrees of each status are known in the art. In anembodiment of the present invention the Gaucher's disease statuscomprises a severe, mild, or healthy Gaucher's disease status.

The term “diagnosing” as used herein, preferably means determining thepresence or the absence of a disease or disorder in a subject and/ordetermining whether a subject is at risk for developing a disease, adisorder or symptoms related to a disease or disorder as well aspredicting a status of a disease.

The term “detecting” in the context of the present invention meansmethods which include detecting the presence or absence of a substancein a sample and/or qualifying the type of said substance. Detecting canbe accomplished by methods known in the art and those further describedherein, including, but not limited to, the direct measurement of theglucosidase enzyme e.g. the sequencing of the gene coding forglucosidase. Any suitable method can be used to detect one or more ofthe biomarkers described herein. These methods include, withoutlimitation, mass spectrometry (e.g. HPLC-MS/MS), fluorescence (e.g.sandwich immunoassay), HPLC-fluorescence or HPLC-UV preferably afterderivatization of free lyso-Gb1.

A biomarker as used herein, preferably is any biological compound, suchas a protein and a fragment thereof, a peptide, a polypeptide, aproteoglycan, a glycoprotein, a lipoprotein, a carbohydrate, a lipid, anucleic acid, an organic or inorganic chemical, a natural polymer, and asmall molecule, which is differentially present in a sample from asubject of one phenotypic status (e.g. having a disease) as comparedwith another phenotypic status (e.g. not having the disease) and whichmay be isolated from, or measured in the sample from the subject.Furthermore, the biomarker can be the entire intact molecule, or it canbe a portion thereof which is preferably detected by mass spectrometricanalysis, an antibody, another protein specifically binding thebiomarker, functional nucleic acids specifically binding the biomarkerand/or a fluorescent label. A biomarker is furthermore considered to beinformative if a measurable aspect of the biomarker is associated with agiven status of the patient, such as a particular status of Gaucher'sdisease. Such a measurable aspect may include, for example, thepresence, absence, or the level of the biomarker in the sample from thesubject and/or its presence as part of a profile of biomarkers. Ameasurable aspect may also be a ratio of two or more measurable aspectsof biomarkers, which biomarkers may or may not be of known identity, forexample. A profile of biomarkers comprises at least two such measurableaspects, where the measurable aspects can correspond to the same ordifferent classes of biomarkers such as, for example, a nucleic acid anda carbohydrate. A biomarker profile may also comprise at least three,four, five, 10, 20, 30 or more measurable aspects. In one embodiment, abiomarker profile comprises hundreds, or even thousands, of measurableaspects. In another embodiment, the biomarker profile comprises at leastone measurable aspect of at least one biomarker and at least onemeasurable aspect of at least one internal standard.

In an embodiment of the method according to the present invention aninternal standard is added to a sample from a subject. It will beacknowledged that by said addition of internal standard, also referredto herein as IS, to the sample, i.e. spiking of the sample, to besubjected to the method according to the present invention, theconcentration of IS in the sample is known and, e.g., by determining thearea under the peak, i.e. the peak area, of the internal standard in,e.g., a HPLC-mass spectrometric chromatogram the relation between a peakarea and a concentration of a substance, e.g. of IS and/or the biomarkerwhich is in the present case free lyso-Gb1, can thus be calculated,e.g., by calculating the ratio of the peak area of free lyso-Gb1 and thepeak area of IS. A person skilled in the art will further acknowledgethat various molecules may be used as an IS. Nevertheless an IS having asimilar chemical structure compared to the molecule such as thebiomarker, e.g. free lyso-Gb1, is preferable. In accordance therewith,the present inventors have in an embodiment chosen lyso-Gb2 whichdiffers from lyso-Gb1 in comprising a further sugar moiety andadditionally is not present as such in nature. In a preferred embodimentthe molecule being the IS can be distinguished from free lyso-Gb1 in themethod of the present invention. In a further preferred embodiment theIS is selected such that a molecule which is ideally not present or rarein nature. In an embodiment of the present invention where the internalstandard is added to a sample from a subject, it is preferred that theIS is added such that it is dissolved in a solvent, e.g. ethanol, priorto said addition to the sample. In a further preferred embodiment thatthe solvent is selected such that said solvent is capable of causingprotein precipitation, preferably is capable of causing the proteinprecipitation step as subject to the method of the present invention.

In some embodiments of the present invention a protein precipitationand/or protein precipitation step is part of the method of the presentinvention. It will be understood that precipitation as used herein,preferably means the formation of a solid in a solution, i.e. forexample the formation of a protein precipitate in a sample, e.g. serum,from a subject. When precipitation, e.g. protein precipitation, occursin a sample, the solid formed is called the precipitate, or whencompacted by a centrifuge, a pellet. The liquid remaining above thesolid is in either case called the supernatant. The present inventioncontemplates different methods of precipitation and/or separating saidsupernatant and said precipitate or pellet, comprising, among others,settling or sedimentation and centrifugation. A person skilled in theart will know further methods for protein precipitation and/or forseparating a supernatant and a protein precipitate, nevertheless saidskilled person will acknowledge that if a method, preferably a method ofthe invention, is applied were precipitated protein will disable adevice such as a column or HPLC-column used in connection with thepresent invention the precipitated protein is preferably separated fromthe solvent and/or the sample.

In some embodiments of the present invention a level of a biomarker ofthe present invention, e.g. free lyso-Gb1, determined by a method of thepresent invention in a sample is compared to a level of the same oranother biomarker of the present invention determined by a method of thepresent invention in another sample, e.g. from the same patient, fromanother patient, from a control and/or from the same or different timepoints, and/or a cut-off level, and/or a level of a control and/or alevel of an IS. In connection therewith “comparing” or “compared to” asused herein, preferably means the mathematical comparison of the two ormore values of the levels of the biomarker(s). It will thus beimmediately evident whether one of said values is higher, lower oridentical if at least two of such values are compared with each other.

In some embodiments of the present invention the level of the biomarkeris also determined in a control. As used herein, a control is preferablya sample from a subject wherein the Gaucher's disease status of saidsubject is known. In an embodiment a control is a sample of a healthypatient. In a further embodiment an amount of said biomarker is added tosaid sample of a healthy patient prior to determining the level of saidbiomarker in said sample of a healthy patient comprising said addedbiomarker with a method of the present invention. In a furtherembodiment the control is a sample from at least one subject having aknown Gaucher's disease status, such known Gaucher's disease statuscomprising severe, mild, or healthy Gaucher's disease status, e.g. acontrol patient. In a further preferred embodiment the control is asample from a subject not being treated for Gaucher's disease. In astill further preferred embodiment the control is a sample from a singlesubject or a pool of samples from different subjects and/or samplestaken from the subject(s) at different time points.

The term “level” or “level of a biomarker” as used herein, preferablymeans the concentration of a substance, preferably of a biomarker of theinvention and more preferably of free lyso-Gb1, within a sample or asubject. It will be understood by a skilled person that in certainembodiments said sample is not necessarily subjected to a method of theinvention as a non-processed sample, the method comprising determining alevel of said biomarker, i.e. said sample may be subjected, e.g. to astep of protein precipitation, separation, e.g. centrifugation and/orHPLC and subsequently subjected to a step of determining the level ofthe biomarker, e.g. using mass spectrometric analysis. It should befurther noted that whenever the term “a” level of a biomarker is used inconnection with a level of the biomarker of the invention which is to bedetermined according to the present invention, “the” level of thebiomarker of the present invention which is to be determined by themethods of to the present invention and which is contained in the samplesubjected to the method(s) of the invention is meant.

The level of a biomarker is different between different statuses ofGaucher's disease, if the mean or median level of the biomarker in thedifferent groups is calculated to be statistically significant. Commontests for statistical significance include, among others, t-test, ANOVA,Wilcoxon, Mann-Whitney, odds ratio and Kruskal-Wallis. Biomarkers, aloneor in combination, provide measures of relative risk that a subjectbelongs to one phenotypic status or another. Therefore, biomarkers ofthe present invention are useful in an embodiment of the presentinvention as markers for disease, therapeutic effectiveness of a drug ora treatment.

The term “determining the level” of a biomarker as used herein,preferably means methods which include quantifying an amount of at leastone substance in a sample from a subject and/or quantifying an amount ofsaid substance contained in a part of the body of the subject, such assaliva, blood, lymph, serum, plasma or liquor and/or quantifying anamount of said substance in the subject, the substance being selectedfrom the group comprising a biomarker.

It will be understood by a person skilled in the art that detectingand/or determining the level of free lyso-Gb1 in a sample from thesubject, thus preferably comprises that Gb1 present in the blood of asubject is not chemically converted, transformed or derivatized suchthat free lyso-Gb1 cannot be detected and/or the level thereof cannot bedetermined separate from and/or apart from Gb1. The person skilled inthe art will acknowledge that Gb1 present in a sample from a subjectwhich is subjected to a step of deacetylation, e.g. by hydrolysis inmethanolic sodium hydroxide, will result in cleavage of the fatty acidmoiety from the Gb1 and thus will undesirably result in a chemicallyconverted, transformed or derivatized form of Gb1 which cannot bedifferentiated from free lyso-Gb1. It is thus the merit of the presentinventors to recognize that free lyso-Gb1 apart from Gb1 is useful in amethod for diagnosing Gaucher's disease.

In a preferred embodiment of the methods of the present invention themethod is for detecting and/or determining the level of free lyso-Gb1 ina sample from a subject, wherein Gb1 present in the sample from thesubject is not subjected to a step resulting in deacetylation of Gb1,preferably is not subjected to a step resulting in cleavage off of afatty acid moiety from the Gb1 contained in the sample. In a furtherpreferred embodiment of the method of the present invention Gb1 presentin the sample from the subject is not chemically converted, transformedor derivatized. In a still further preferred embodiment of the method ofthe present invention free lyso-Gb1 present in the sample from thesubject is separated from Gb1 present in the sample from the subjectprior to a step that would result in cleavage of a fatty acid moietyfrom the Gb1 and/or prior to a step in which Gb1 is chemicallyconverted, transformed or derivatized. In a still further preferredembodiment a step of detecting and/or determining the level of abiomarker in a sample from the subject, wherein the biomarker is freelyso-Gb1, is performed subsequent to separation using HPLC byapplication of mass spectrometric analysis.

A subject is considered to be a healthy subject with regard to Gaucher'sdisease, if the subject does not suffer from symptoms associated withGaucher's disease. Moreover in an embodiment of the methods of theinvention a subject will be considered to be healthy if it has nomutation of the functional parts of the cerebrosidase gene and/or nomutation of the cerebrosidase gene resulting in a reduction of ordeficiency of the enzyme glucocerebrosidase or the activity thereof,resulting in symptoms associated with Gaucher's disease. Said mutationswill be detected if a sample from the subject is subjected to a genetictesting for such mutations as described herein. In a further embodimentof the present invention a sample from a healthy subject is used as acontrol sample or as a blank matrix in the methods of the presentinvention. A blank matrix as referred to herein, is preferably a samplefrom a healthy subject. Nevertheless it will be understood that such ablank matrix may contain a native level of free lyso-Gb1.

In an embodiment of the present invention the level of a biomarker isindicative for the subject for suffering from or for being at risk fordeveloping a disease or disorder. The level of the biomarker determinedby the method according to the present invention is compared to acontrol level of the biomarker, wherein the result of said comparisonallows for diagnosing a disease.

More specifically, comparing the level of the biomarker in the samplefrom the subject to the control level of the biomarker comprisescomparing the level of the biomarker in the sample from the subject to acut-off level, wherein if a level of the biomarker in the sample fromthe subject is elevated, increased or higher compared to the cut-offlevel, this is indicative that the subject is suffering from or is atrisk for developing Gaucher's disease and/or, wherein if a level of thebiomarker in the sample from the subject is decreased or lower comparedto the cut-off level this is indicative that the subject is notsuffering from or is not at risk for developing Gaucher's disease. It isalso within the present invention that comparing the level of thebiomarker in the sample from the subject to a control level allows fordetermining the severity of Gaucher's disease, wherein if a level of thebiomarker in the sample from the subject is elevated, increased orhigher compared to the control level that is indicative that the subjectis suffering from or is at risk for developing Gaucher's disease of amore severe status or progression; and wherein if a level of thebiomarker in the sample from the subject is decreased or lower comparedto the control level that is indicative that the subject is sufferingfrom or is at risk for developing Gaucher's disease of a less severestatus or progression. In a further embodiment of the present inventionthat comparing the level of the biomarker in the sample from the subjectto the control level comprises comparing a level of the biomarker insaid subject to a level of the biomarker detected in a sample from acontrol, wherein if a level of the biomarker in the sample from thesubject is elevated, increased or higher compared to the control samplethis is indicative that the subject is suffering from and/or is at riskfor developing Gaucher's disease; and/or a level of the biomarker in thesample from the subject is elevated, increased or higher compared to thecontrol sample this is indicative that the subject is suffering from oris at risk for developing Gaucher's disease of a more severe status orprogression. Said control preferably is selected from the groupcomprising healthy subjects, subjects suffering from Gaucher's diseaseor being at risk of suffering from Gaucher's disease symptoms, subjectsbeing positively tested for a mutation or a combination of mutations ofthe cerebrosidase gene, wherein the mutation or the combination ofmutations of the cerebrosidase gene are indicative for a perspective ofthe subject to develop Gaucher's disease of a more severe or less severestatus or progression. In a further embodiment of the present inventionthat a control level is determined in a sample from a control, whereinoptionally free lyso-Gb1 is added to the sample from the control in aspecific quantity prior to determining the level of free lyso-Gb1 in thesample from the control.

It is the merit of the present inventors that a method for diagnosingGaucher's disease in a subject could be established wherein the methodcomprises detecting a biomarker in a sample from a subject, wherein thebiomarker is free lyso-Gb1, preferably further comprising determining alevel of the biomarker in the sample from the subject, and morepreferably further comprising comparing the level of the biomarker inthe sample from the subject to a cut-off level, which shows highsensitivity, i.e. a sensitivity of at least 99.0%, 99.1%, 99.2%, 99.3%,99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% and high specificity ofat least 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%,99.9% or 100%. In a further embodiment of the present invention that themethods according to the present invention allow for diagnosingGaucher's disease in a subject independent from a progression status ofGaucher's disease in the subject. More specifically, the methods of thepresent invention allow for diagnosing Gaucher's disease in a subjecthaving an early status of Gaucher's disease as well as in a subjecthaving an advanced or progressed status of Gaucher's disease.

The power of a method to correctly diagnose Gaucher's disease, iscommonly measured as the sensitivity of the method, the specificity ofthe method or the area under a receiver operated characteristic curve(also referred to herein as “ROC curve”). An ROC curve is a plot of thetrue positive rate against the false positive rate for the differentpossible cut-off levels of a diagnostic method. An ROC curve shows therelationship between sensitivity and specificity. Sensitivity is thepercentage of true positives that are predicted by a test to bepositive, while specificity is the percentage of true negatives that arepredicted by a test to be negative. An ROC-curve provides thesensitivity of a test as a function of 1-specificity. The greater thearea under the ROC-curve the more powerful the predictive value of thetest. Accordingly, an increase in sensitivity will be accompanied by adecrease in specificity. The closer the curve follows the left axis andthen the top edge of the ROC space, the more accurate the test.Conversely, the closer the curve comes to the 45-degree diagonal of theROC graph, the less accurate the test. Therefore, the area under the ROCis a measure of test accuracy. The accuracy of the test depends on howwell the test separates the group being tested into those with andwithout the disease in question. An area under the curve (also referredto herein as “AUC”) of 1 represents a perfect method, while an area of0.5 represents a less useful method. Thus, preferred diagnostic methodsof the present invention have an AUC greater then 0.50, more preferredmethods have an AUC greater than 0.9 and most preferred methods have anAUC greater than 0.998.

Other useful and suitable measures for the utility of a method arepositive predictive value and negative predictive value. A positivepredictive value is the percentage of actual positives that test aspositive. A negative predictive value is the percentage of actualnegatives that test as negative.

Methods for qualifying Gaucher's disease status in a subject that usebiomarkers of the prior art, e.g. chitotriosidase and/or CCL18 show asensitivity and specificity typically not more than 90%.

A person skilled in the art will acknowledge that although thespecificity and the sensitivity of the methods according to the presentinvention are as high as described above and were determined asdescribed in the Examples hereinafter, individual cases may not beexcluded where a patient having Gaucher's disease will be tested falsenegative or where a patient not having Gaucher's disease will be testedfalse positive with a method of the invention. Taking said cases intoaccount while determining the specificity and the sensitivity of themethod according to the present invention, the specificity and thesensitivity will be lower than the above described values. Nevertheless,the person skilled in the art will also acknowledge that such highspecificity and such high sensitivity as has been outlined above hasnever been described before for a method for diagnosing Gaucher'sdisease. Therefore it is important to note that although the sensitivityand the specificity of the method of the present invention may vary ifpatient collectives other than the one reported in the Example part,e.g. varying in number of patients, will are subject to the methods ofthe present invention, it is the firm belief of the inventors that nomethod known in the prior art using biomarkers will achieve a higherspecificity and a higher sensitivity compared to the methods accordingto the present invention. This is especially true since the limit ofdetection of the methods of the present invention allows for determiningthe level of free lyso-Gb1 in many healthy subjects. Accordingly, adiseased subject tested false negative applying the methods of thepresent invention is tested false negative for the reason that a levelof the biomarker in a sample from said false negative tested diseasedsubject is as high as the level of the biomarker in a sample from ahealthy subject. In particular it is important to note that said falsenegative tested subject is not tested negative for the reason that thelevel of the biomarker was too low to be determined by the method of thepresent invention.

A “limit of detection” of a substance such as free lyso-Gb1, as usedherein, preferably is a level of the substance determined by a methodfor determining a level of the substance, wherein a level less then orlower then said limit of detection cannot be determined by said method.It is thus immediately clear that a “cut-off level” and a “limit ofdetection”, as used herein, are preferably not necessarily identical,although both reflect a certain level of a substance, e.g. of abiomarker of the present invention. It will be immediately understoodthat in contrast to a cut-off level will be selected preferably suchthat selectivity and sensitivity of the method are as high as possible.In contrast thereto a limit of detection represents an absolute level ofthe biomarker of the present invention which reflects the minimum levelof biomarker which can be detected with a method for determining thelevel of said biomarker. It is thus immediately clear that a limit ofdetection depends on the method for determining a level of a substanceand on the substance the level of which is to be determined by themethod. A skilled person will immediately understand that a high limitof detection, e.g. higher than an ideal cut-off level would possiblyresult in a low sensitivity of the method since the percentage of truepositives that are predicted by a test to be positive also depends onwhether a level of the biomarker may be determined for said truepositives. In other words, if the limit of detection is higher than anideal cut-off level, true positives having a level of the biomarkerslightly higher than the cut-off level may not be distinguished fromtrue negatives having a level of the biomarker lower than the cut-offlevel since no level of the biomarker may be determined for both truepositives having a level of the biomarker slightly higher than thecut-off level and negatives having a level of the biomarker lower thanthe cut-off level. It is thus immediately clear that a low limit ofdetection is of advantage. It is therefore also the merit of theinventors to show that a lower limit of detection allows for a methodfor diagnosing Gaucher's disease in a subject comprising a step ofdetermining a level of a biomarker present in the sample with higherselectivity and sensitivity. An “ideal cut-off level” as used herein,preferably is the cut-off level as described herein the method usingsaid ideal cut-off level has the highest selectivity and sensitivity.

It is an embodiment of the methods according to the present invention tocomprise a step of validating said method by diagnosing a disease ordisorder, preferably Gaucher's disease in a subject by the method of thepresent invention; a step of diagnosing the disease or disorder,preferably Gaucher's disease, in a subject by a genetic testing,comprising sequencing of a gene, preferably sequencing of a gene amutation of which is known to the one skilled in the art to cause thedisease or disorder, more preferably sequencing the cerebrosidase genein case of Gaucher's disease; and comparing the results of said methodand said genetic testing. A healthy subject as used herein, preferablyis considered to be healthy with regard to a disease or disorder if saidsubject is not suffering from symptoms associated with said disease ordisorder and if the result of a genetic testing reveals no mutations ofa gene a mutation of which is known to the one skilled in the art tocause the disease or disorder. A healthy subject also is understood tobe a subject being positively tested for not having Gaucher's disease.

The term “qualifying Gaucher's disease status” in a subject as usedherein, preferably means a classification of a subject's biomarkerprofile selected from the group comprising to identify or detect thepresence or absence of Gaucher's disease in the subject, to predict theonset of or the risk for developing of Gaucher's disease in the subject,to determine the course of Gaucher's disease in a subject, to determineand/or predict the severity of Gaucher's disease in a subject, todetermine whether a subject suffers from an early status of Gaucher'sdisease or an advanced or progressed status of Gaucher's disease or todetermine whether a level of a biomarker in a subject has significantlychanged over time.

The term “managing subject treatment” or “subject management” as usedherein, preferably refers to the behavior of the clinician or physiciansubsequent to the determination of Gaucher's disease status. Forexample, if the result of the method according to the present inventionis inconclusive or there is reason that confirmation of status isnecessary, the physician may order new tests, such as testing for thefunction of the glucocerebrosidase and/or sequencing of the gene codingfor the glucocerebrosidase. Alternatively, if the status indicates thattreating for Gaucher's disease is appropriate, the physician mayschedule the subject for treating for Gaucher's disease. Likewise, ifthe status is negative or if the results show that treatment has beensuccessful, no further management may be necessary. Nevertheless aperson skilled in the art will immediately acknowledge that besides genetherapy any therapy applied, e.g. ERT and/or SRT has to be appliedlifelong to a Gaucher's disease patient. Furthermore it is an embodimentof the present invention that managing subject treatment comprisestitrating of a dose of a drug applied as a treatment for Gaucher'sdisease, e.g. units of recombinant enzyme applied in ERT, administeredto a patient. In some embodiments of the methods of the presentinvention wherein a level of a biomarker present in a sample from asubject is determined at several points in time, or is compared to otherlevels of the biomarker, a cut-off level and/or a level of saidbiomarker in a control, a skilled person will apply or not apply atherapy, or amend a therapy already applied in order to treat or not totreat, or to continue treating Gaucher's disease.

It is within the present invention that a skilled person will apply adosage and/or maintain a dosage or amend a dosage, e.g. apply a dosageor a higher dosage, i.e. elevate a dosage, if such a comparison of thelevel of a biomarker shows e.g. that the level of said biomarker ishigher than for example, a cut-off level, i.e. the patient is diagnosedto have Gaucher's disease; or that a level determined in the samepatient earlier in time is lower or the same, i.e. a therapy applied isnot sufficient, i.e. does not result in a decrease in the level. On theother hand skilled person will apply or not apply a dosage or maintainor reduce a dosage, e.g. apply no dosage or a lower dosage, i.e.decrease a dosage, if such a comparison of the level of a biomarkershows e.g. that the level of said biomarker is lower than for example, acut-off level, i.e. the patient is diagnosed not to have Gaucher'sdisease; or that a level determined in the same patient earlier in timeis higher, i.e. a therapy applied is sufficient, i.e. does result in adecrease in the level. In an embodiment of the present invention arelatively high level of free lyso-Gb1 based on such a comparison isindicative for applying a high dosage of recombinant enzyme applied inERT and/or a relatively low level of free lyso-Gb1 based on such acomparison is indicative for applying a low dosage of recombinant enzymeapplied in ERT. Nevertheless it will also be immediately understood thata skilled person will consider a patient's history, i.e. a skilledperson managing subject treatment of a patient suffering from Gaucher'sdisease and being treated such that a level of biomarker is lower than acut-off level, for example, will not decide to stop treatment ratherthan decrease a dosage and increase the time between furtherapplications of the methods of the present invention.

The course of Gaucher's disease may be determined by the methodaccording to the present invention by determining a level of thebiomarker in the sample from the subject at different time points in thecourse of the disease. It is important to note that a single applicationof a method for diagnosing Gaucher's disease according to the presentinvention allows for diagnosing Gaucher's disease and in certainembodiments comprises a step of managing subject treatment based on thediagnosis of whether the subject is suffering from or for being at riskfor developing Gaucher's disease. If a subject a sample of which is thussubjected to the method of the present invention is tested positive forsuffering from or to be at risk for developing Gaucher's disease askilled clinician will know how to decide concerning managing subjecttreatment, i.e. how the subject will be treated, e.g. applying a certaindose of enzyme in relation to an ERT. It will be immediately understoodthat independent of the decision of a skilled clinician on how to managesubject treatment the skilled clinician may decide for at least oneadditional application of the method according to the present inventionon a later time point. It is thus an embodiment of the present inventionthat the levels of the biomarker determined at the different timepoints, wherein different time points means at least two time points,may be compared. Without wishing to be bound by any theory the presentinventors have found that the level of the biomarker of the presentinvention in samples form one particular patient may be correlated tothe severity of the disease in said patient at the time point the samplefrom the patient is taken. It will be thus immediately understood thatan elevated level of the biomarker determined in the sample of a latertime point compared to the level of the biomarker determined in thesample of an earlier time point is indicative for a more severe statusof the subject at the later time point compared to the status of thesubject at the earlier time point. A decreased level of the biomarkerdetermined in the sample of a later time point compared to the level ofthe biomarker determined in the sample of an earlier time point isindicative for a less severe status of the subject at the later timepoint compared to the status of the subject at the earlier time point.Accordingly, in one aspect the present invention provides a method fordetermining the course of Gaucher's disease in a subject comprising thestep of determining at several points in time a level of a biomarkerpresent in a sample from the subject, wherein the biomarker is freelyso-Gb1. In a further aspect the invention concerns a method fordetermining the effectiveness of at least one treatment applied to asubject being positively tested for suffering from or being at risk fordeveloping Gaucher's disease comprising the step of determining atseveral points in time a level of a biomarker present in a sample fromthe subject, wherein the biomarker is free lyso-Gb1. It will beimmediately understood by a person skilled in the art that the methodsof the present invention thus allow for selecting a therapy and/oradjusting the doses and/or dosage of a selected therapy based on theresults of the method of the invention. If for example the subject isscheduled for treating for Gaucher's disease the method for diagnosingGaucher's disease in a subject according to the present invention may beapplied every 3 months and levels of the biomarker thus determined willbe compared in order to determine the effectiveness of the treatment(s)and/or therapy/therapies applied to the subject. If the subject reachesa status, wherein a stable level of the biomarker is maintained overtime the frequency of application of the method for diagnosing Gaucher'sdisease in a subject according to the present invention may be reducedto every 6 month. If the dosage of the therapy is changed, e.g. theunits of recombinant enzyme applied in ERT are reduced or increased, thefrequency of application of the method for diagnosing Gaucher's diseasein a subject according to the present invention may be set back to every3 month. By comparison of the determined levels of the biomarker in thesamples from the subject the skilled physician will recognize whetherthe level of the biomarker increases, decreases or whether a stablelevel of the biomarker is maintained over time. Accordingly, the skilledphysician may decide to reduce the dosage of the therapy, e.g. the unitsof recombinant enzyme applied in ERT; to increase the dosage of thetherapy; or to maintain the dosage of the therapy according to thecomparison of the levels of the biomarker determined with the methodaccording to the present invention. A reduction of about 60% of thelevel of free lyso-Gb1 within a period of 12 month is indicative for asuccessful therapy for Gaucher's disease, wherein reduction as usedherein, preferably means that the level of free lyso-Gb1 determined bythe method of the present invention determined at the end of a timeperiod is compared to the level of free lyso-Gb1 determined by themethod of the present invention determined at the beginning of said timeperiod. Accordingly the skilled physician may decide to reduce thedosage of the applied therapy or to maintain the dosage of the therapy.If the reduction of the level of free lyso-Gb1 is significantly weakerthe skilled physician may decide to increase the dosage of the therapy.It is also a merit of the present inventors to have recognized that thereduction of the level of free lyso-Gb1 correlates with theeffectiveness of a therapy. The stronger the reduction of the level ofthe free lyso-Gb1 within a time period, e.g. 12 months, the moresuccessful is a therapy, such as for example ERT, SRT or a chaperonebased therapy. It is thus a further embodiment of the present inventionthat the method of the present invention is for comparing theeffectiveness of a therapy or of at least two therapies applied to asubject.

A person skilled in the art thus will acknowledge that the progression,i.e. course of Gaucher's disease, as well as the effectiveness of atherapy in a single subject can be monitored by frequent determining ofthe level of free lyso-Gb1 in samples from the subject.

In a further aspect the invention concerns a method for determining theeffectiveness of at least one treatment applied to a subject beingpositively tested for suffering from or being at risk for developingGaucher's disease comprising the step of determining at several pointsin time a level of a biomarker present in a sample from the subject,wherein the biomarker is free lyso-Gb1. In connection with what has beenoutlined above in relation to managing subject treatment a personskilled in the art will immediately understand that the effectiveness ofone treatment or the combination of at least two treatments may becompared applying the methods of the present invention. Thus it ispossible to test and compare several new drugs, dosage forms, dosages ortreatments for Gaucher's disease by the method of the present invention.

It is an embodiment of the present invention that the method fordiagnosing Gaucher's disease according to the present invention isindependent of whether the subject has or has not been previouslytreated for Gaucher's disease. Thus the sample from the subject may be asample from a subject who has been previously treated for Gaucher'sdisease as well as a sample from a subject who has not been previouslytreated for Gaucher's disease. It is thus a further embodiment of thepresent invention that the method of the present invention comprises astep of managing subject treatment and/or determining a level of thebiomarker in the sample from the subject after subject management. Saidsubject treatment can be based on the diagnosis of whether the subjectis suffering from or for being at risk for developing Gaucher's disease;on the detection of the biomarker in a sample from the subject aftersubject management; or on the determining of the level of the biomarkerin the sample from the subject after subject management. Nevertheless aperson skilled in the art will understand that a sample of some patientsnot having Gaucher's disease or of some patients being successfullytreated for Gaucher's disease will show a level of free lyso-Gb1 lowerthan the limit of detection.

Without wishing to be bound by any theory the present inventors assumethat the level of free lyso-Gb1 present in a sample from a subjectfurther correlates with the severity of the disease in a subjectsuffering from Gaucher's disease. In connection therewith the presentinventors found by evaluating the results provided herein (e.g. shown inFIG. 4 herein) that although, in principle, the level of free lyso-Gb1is different in particular individuals, and more specifically may bedifferent in particular individuals having the same mutation(s), thatthe higher is a level of free lyso-Gb1, the higher is the severity of acourse of Gaucher's disease in terms of a statistical mean according toa clinical score. Thereby the level of free lyso-Gb1 correlates with theseverity of Gaucher's disease in that in patients being positivelytested for distinct mutations of the glucocerebrosidase gene being knownto generally causes a mild (e.g. N370S mutation) or a more severe (e.g.L444P mutation) course of Gaucher's disease, a level of free lyso-Gb1determined in said patients statistically correlated with the severitygenerally related to such mutation.

Thus a further embodiment of the different aspects of the presentinvention concerns a method for determining the severity of Gaucher'sdisease in a subject comprising a step of

-   -   a) determining a level of the biomarker present in a sample from        the subject wherein the biomarker is free lyso-Gb1 and a step of    -   b) determining the severity of Gaucher's disease, e.g. by        comparing the level of free lyso-Gb1 in a subject preferably        determined by a method of the present invention to a clinical        score.

In connection therewith it is important to note that if a level of freelyso-Gb1 is determined in samples from the patients suffering fromGaucher's disease showing the L444P mutation upon sequencing of thecerebrosidase gene (homozygous and compound heterozygous) subjected to amethod of the present invention a mean-level of free lyso-Gb1 is higherthan the mean-level of the free lyso-Gb1 determined in samples from thepatients suffering from Gaucher's disease showing the N370S mutationupon sequencing of the cerebrosidase gene, applying the same method(FIG. 4). Mutation L444P is known to cause a more severe course ofGaucher's disease—this is especially true in case the subject ishomozygous as to said mutation. Corresponding to that a highermean-level of free lyso-Gb1 is determined in the homozygous compared tothe homozygous N370S mutation (194 ng/ml and 159 ng/ml, respectively,see FIG. 4). Moreover patients having a compound heterozygous L444Pmutation have a significant lower free lyso-Gb1 level than homozygousones (89 ng/ml and 45.4 ng/ml, respectively). A person skilled in theart will know clinical scores to categorize the severity of Gaucher'sdisease or symptoms or an entirety of symptoms thereof. It is thus anembodiment of the method of the present invention that the course ofGaucher's disease in a patient is predicted and more particularly theseverity of Gaucher's disease is determined based on the level of thebiomarker determined according to the method of the present invention.

It is an embodiment of the present invention that levels ofchitotriosidase determined in patients not having a mutation of thechitotriosidase gene, in particular not having a 24-bp duplication asdescribed herein, serve as a basis for correlating the severity ofGaucher's disease in that a mean-level of chitotriosidase determined ina sample from said patients as described herein is correlated to aseverity of Gaucher's disease. Thus, for example a level ofchitotriosidase below 200 nmolMU/h/ml is correlated with a Gaucher'sdisease status of a patient not suffering from Gaucher's disease. Inconnection therewith it is important to note that a patient treated forGaucher's disease may also exhibit a level of chitotriosidase below 200nmolMU/h/ml. A level of more than 2000 nmolMU/h/ml is correlated to a“fullblown” or “severe” Gaucher's disease status and a level ofchitotriosidase from 200 to 2000 nmolMU/h/ml is correlated to a “mild”Gaucher's disease status.

In connection therewith is important to note that a level ofchitotriosidase from 200 to 1000 nmolMU/h/ml may also be found in asample from a subject suffering from another LSD such as Niemann-Picktype C or Krabbe's disease, thereby rendering the use of chitotriosidasefor diagnosing Gaucher's disease unsuitable. Therefore, theconsiderations outlined above in connection with the use of a level ofchitotriosidase for correlation with the severity of Gaucher's diseasetypically apply only for patients wherein the presence or absence ofGaucher's disease and/or other LSD known to show elevated levels ofchitotriosidase was proven by mutational analysis.

If a level of free lyso-Gb1 is determined according to the methods ofthe present invention in said patients not having a mutation of thechitotriosidase gene, in particular not having a 24-bp duplication asdescribed herein, said level of free lyso-Gb1 determined in a samplefrom each of said patients is correlated to the chitotriosidase level ofsaid patients and/or to the grade of severity of Gaucher's diseaseand/or status of Gaucher's disease of said patient. Thus a grade ofseverity of Gaucher's disease and/or status of Gaucher's disease,comprising healthy, mild and severe is determined and more preferably iscorrelated to levels of chitotriosidase and/or ranges of levels ofchitotriosidase as outlined above.

A person skilled in the art will acknowledge that a level of thebiomarker of the present invention determined in a sample from a subjectwherein said level of the biomarker is correlated with the severity ofGaucher's disease as described above, will be indicative for applying acertain therapy and/or dose or dosage of said therapy. For example, ifthe level of the biomarker in determined according to the methods of theinvention is correlated with “severe” or “fullblown” Gaucher's diseasestatus the subject is scheduled for treatment of Gaucher's disease andthe method for diagnosing Gaucher's disease in a subject according tothe present invention may be applied every 3 months and levels of thebiomarker thus determined will be compared in order to determine theeffectiveness of the treatment(s) and/or therapy/therapies applied tothe subject. If the subject reaches a status, wherein the level of thebiomarker is correlated with a “mild” Gaucher's disease or wherein astable level of the biomarker is maintained over time the frequency ofapplication of the method for diagnosing Gaucher's disease in a subjectaccording to the present invention may be reduced to every 6 month.

In another aspect the present invention is related to a method ofdetermining the effectiveness of a composition for the treatment ofGaucher's disease. Such method may comprise the steps of determining alevel of free lyso-Gb1 in a subject having Gaucher's disease;administering to said subject said compound in an amount sufficient todetermine the effectiveness of said compound; re-determining the levelof free lyso-Gb1 in said subject; comparing the level of free lyso-Gb1determined before and after administering said composition, wherein alower level of free lyso-Gb1 determined after administering saidcomposition compared to the level of free lyso-Gb1 determined afteradministering said composition indicates the effectiveness of saidcompound for treating Gaucher's disease.

The present invention is now further illustrated by the followingfigures and examples from which further features, embodiments andadvantages may be taken.

More specifically,

FIG. 1A is a boxplot indicating levels of free lyso-Gb1 in ng/ml plasma;

FIG. 1B is a boxplot indicating levels of free lyso-Gb1 in ng/ml plasmagrouped by gender of the subjects;

FIG. 2A is a graph showing receiver operating characteristics (ROC)curves of free lyso-Gb1 and chitotriosidase;

FIG. 2B is a graph showing receiver operating characteristics (ROC)curves of free lyso-Gb1 and CCL18;

FIG. 3A is a diagram showing free lyso-Gb1 in ng/ml plasma as a functionover time for a total of 20 German Gaucher's disease patients;

FIG. 3B is a diagram showing free lyso-Gb1 in ng/ml plasma as a functionover time for a total of 24 non-treated Gaucher's disease patients (10German, 14 Israeli patients);

FIG. 3C is a diagram showing free lyso-Gb1 in ng/ml plasma as a functionover time for a total of 9 Israeli Gaucher's disease patients before andafter start of therapy;

FIG. 3D is a diagram showing regression based values of free lyso-Gb1 inng/ml plasma as a function over time for Israeli and German Gaucher'sdisease patients before and after start of therapy;

FIG. 4 is a table showing the median level of free lyso-Gb1 for twofrequent mutations;

FIG. 5A is an HPLC-mass spectrometric chromatogram displaying peakintensity of free lyso-Gb1 and IS of a healthy subject;

FIG. 5B is an HPLC-mass spectrometric chromatogram displaying peakintensity of free lyso-Gb1 and IS of a Gaucher's disease patient;

FIG. 5C is an HPLC-mass spectrometric chromatogram displaying peakintensity of free lyso-Gb1 and IS of a Gaucher's disease patient;

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a boxplot indicating levels of free lyso-Gb1; the y-axisdemonstrates the logarithmised levels of free lyso-Gb1 in ng/mldetermined in plasma of Patients by the method according to the presentinvention, wherein the x-axis depicts groups of patients, which havebeen grouped as described in Example 2. The boxplot represents the25^(th) and 75^(th) percentile of each group of patients by the bottomand top of the box, respectively; the band near the middle of the boxrepresents the 50^(th) percentile (i.e. the median) of each group; Thewhiskers represent one standard deviation above and below the mean ofthe data; Any data not included between the whiskers is shown as anoutlier with a small circle or star. The horizontal line represents thecut-off level of 5 ng/ml.

FIG. 1B is a boxplot indicating the levels of free lyso-Gb1 as depictedin FIG. 1A additionally grouped by gender of the subjects; the y-axisrepresents the logarithmised levels of free lyso-Gb1 in ng/ml determinedin plasma of patients by the method according to the present invention,wherein the x-axis represents groups of patients, which have beengrouped as described in Example 2 and additionally by the gender of thepatients. The boxplot represents the 25^(th) and 75^(th) percentile ofeach group of patients by the bottom and top of the box, respectively;the band near the middle of the box represents the 50^(th) percentile(e.g. the median) of each group; The whiskers represent one standarddeviation above and below the mean of the data; Any data not includedbetween the whiskers is shown as an outlier with a small circle or star.The horizontal line represents the cut-off level of 5 ng/ml.

FIG. 2A is a graph showing receiver operating characteristics (ROC)curves of free lyso-Gb1 and chitotriosidase; the x-axis represents“1-specificity” and the y-axis represents the sensitivity. Free lyso-Gb1demonstrates a 100% sensitivity and 100% specificity, whereinchitotriosidase has at the best a sensitivity of 0.9591 or 95.91%,respectively.

FIG. 2B is a graph showing receiver operating characteristics (ROC)curves of free lyso-Gb1 and CCL18; the x-axis represents “1-specificity”and the y-axis represents the sensitivity. Free lyso-Gb1 demonstrates asensitivity of 100% and a specificity of 100%, wherein CCL18 has at thebest a sensitivity of 0.8658 and 86.58%, respectively.

FIG. 3A The y-axis represents levels of free lyso-Gb1 as a function overtime determined by the method according to the present invention inng/ml of plasma of 20 German Gaucher's disease patients which weresubjected to therapy, more precisely ERT, during the course of thestudy. Each curve and each patient number, respectively, representslevels determined in plasma collected from the same patient at differenttime points as indicated on the x-axis. The x-axis represents the timepoints of plasma collection, wherein time point zero indicates the firstmeasure under therapy for each patient. For the analysis of the changeof the level of free lyso-Gb1 over time in Gaucher's disease patients asdescribed in Example 3 non aggregated data was used for those patientsfor which more than one blood sample has been analysed.

FIG. 3B is a diagram showing free lyso-Gb1 as a function over timedetermined by the method according to the present invention in ng/ml ofplasma of a total of 24 non-treated Gaucher's disease patients (10German, 14 Israeli patients); Non-treated as used herein, preferablymeans that no treatment, e.g. enzyme replacement therapy, has beenapplied with regard to Gaucher's disease. For the analysis of the changeof the level of free lyso-Gb1 over time in Gaucher's disease patients asdescribed in Example 3 non aggregated data was used for those patientsfor which more than one blood sample has been analysed.

FIG. 3C is a diagram showing free lyso-Gb1 as a function over time withthe free lyso-Gb1 being determined by the method according to thepresent invention in ng/ml of plasma of a total of 9 Israeli Gaucher'sdisease patients during time before and after the start of the therapy.The x-axis indicates the time in month, wherein “0” indicates the firstpoint in time after start of therapy. The curve labeled with “overall”depicts the regression based values of free lyso-Gb1.

FIG. 3D is a diagram showing the regression based values of freelyso-Gb1 as a function over time determined by the method according tothe present invention in ng/ml of plasma of a Israeli and GermanGaucher's disease patients during time before and after the start of thetherapy. The x-axis indicates the time in months, wherein “0” indicatesthe first point in time after start of therapy. The curve labeled with“overall” depicts the regression based values of free lyso-Gb1.

FIG. 4 is a table showing the median level of free lyso-Gb1 in patientspositively tested for one of two frequent mutations of theglucocerebrosidase gene, namely N370S and L444P in the homozygous aswell as in the compound heterozygous situation, wherein compoundheterozygosity is the condition of having two heterogeneous recessivealleles at a particular locus that can cause genetic disease in aheterozygous state. A person skilled in the art will acknowledge thatpatients having a mutation L444P of the glucocerebrosidase gene alsoface a more malignant prognosis, which is particularly true in thehomozygous situation. Accordingly, it is an embodiment of the presentinvention that the method according to the present invention comprisesdetermining the severity of Gaucher's disease. Said determining theseverity of Gaucher's disease comprises determining a level of thebiomarker, preferably free lyso-Gb1, present in the sample from thesubject and/or comparing said level of said biomarker determined insamples from subjects having different mutations of theglucocerebrosidase gene and/or having no mutation of theglucocerebrosidase gene. The present inventors have found that in asample from a patient being positively tested for having a homozygousL444P mutation of the cerebrosidase gene the level of free lyso-Gb1determined by the method according to the present invention is about 194ng/ml and is elevated compared to the level of free lyso-Gb1 determinedin a sample from a patient being positively tested for having ahomozygous N370S mutation of the cerebrosidase gene, wherein the levelof free lyso-Gb1 determined by the method according to the presentinvention is about 159 ng/ml). The inventors have also found that in asample from a patient being positively tested for having a compoundheterozygous L444P mutation the level of free lyso-Gb1 determined by themethod according to the present invention is 89 ng/ml and issignificantly lower compared to the level of free lyso-Gb1 determined bythe method according to the present invention in a sample from a patientbeing positively tested for having a homozygous L444P mutation whereinthe level of free lyso-Gb1 determined by the method according to thepresent invention is about 45.4 ng/ml. Without wishing to be bound bytheory the present inventors believe that the level of free lyso-Gb1 ina sample from a subject determined by a method of the present inventionis indicative for the severity of Gaucher's disease. It is thus afurther embodiment of the present invention that the method of thepresent invention is for determining the effectiveness of at least onetreatment applied to a subject being positively tested for sufferingfrom and/or being at risk for developing Gaucher's disease. The numbersdepicted in brackets indicate the ranges of concentration measured inthe respective patient group. IQR means interquartile range. Allpatients that were subjected to a therapy for Gaucher's disease weresubjected to ERT.

FIG. 5A is an HPLC-mass spectrometry chromatogram displaying peakintensity in cps of free lyso-Gb1 and IS of a sample from a healthysubject as a function over the retention time in minutes. The retentiontime of a substance as used herein, preferably is depicted on the x-axisand is the elapsed time between the time of injection of a solute, e.g.a biomarker according to the present invention and/or an internalstandard, and the time of elution of the peak maximum of said solute. Aperson skilled in the art will acknowledge that the retention time of asubstance according to the herein described methods is a uniquecharacteristic of said solute and can be used for identificationpurposes. Internal Standard working solution comprising Lyso-Gb2 as aninternal standard was added to the sample as described in Example 1. Itis important to understand that by said addition of IS to the sample,i.e. spiking of the sample, to be subjected to the method according tothe present invention, the concentration of IS in the sample is knownand by determining the area under the peak, i.e. the peak area, of theinternal standard in said HPLC-mass spectrometric chromatogram therelation between a peak area and a concentration of a substance, e.g. ofIS and/or a biomarker thus can be calculated. More precisely, a personskilled in the art will acknowledge that a peak area of a substancedepicted in an HPLC-mass spectrometric chromatogram, such as theHPLC-mass spectrometric chromatogram depicted in FIG. 5A, FIG. 5B orFIG. 5C, represents a measure for an amount of said substance subjectedto an HPLC-mass spectrometric analysis. Moreover, a person skilled inthe art will be able to calculate the amount of the substance in asample from a subject subjected to an HPLC-mass spectrometric analysis,e.g. the amount of free lyso-Gb1 in a sample subjected to the method ofthe present invention, using a ratio of the peak area of free lyso-Gb1,the amount of which is to be determined by said method and the peak areaof IS, e.g. free lyso-Gb2; as well as calibration curves generated withsaid method and said free lyso-Gb1 and/or IS. Accordingly, this allowssubsequently for determining a level of free lyso-Gb1.

FIG. 5B is an HPLC-mass spectrometry chromatogram displaying peakintensity of free lyso-Gb1 and IS of a sample from a Gaucher's diseasepatient, wherein a level of 17.1 ng/ml free lyso-Gb1 was determinedaccording to the method of the present invention as essentiallydescribed in Example 1. Comparing said level of the biomarker in thesample from the subject to a cut-off level of 5 ng/ml, which has beenselected such that a sensitivity for diagnosing Gaucher's disease in asubject according to the methods of the present invention is 100% andthat a specificity for diagnosing Gaucher's disease in a subjectaccording to the methods of the present invention is 100%, an elevatedlevel of the biomarker in the sample from the subject compared to thecut-off level is indicative for the subject for suffering from Gaucher'sdisease.

FIG. 5C is an HPLC-mass spectrometry chromatogram displaying peakintensity of free lyso-Gb1 and IS of a sample from a Gaucher's diseasepatient, wherein a level of 319 ng/ml free lyso-Gb1 was determinedaccording to the method of the present invention as essentiallydescribed in Example 1. Comparing said level of the biomarker in thesample from the subject to a cut-off level of 5 ng/ml, which has beenselected such that a sensitivity for diagnosing Gaucher's disease in asubject according to the methods of the present invention is 100% andthat a specificity for diagnosing Gaucher's disease in a subjectaccording to the methods of the present invention is 100%, an elevatedlevel of the biomarker in the sample from the subject compared to thecut-off level is indicative that the subject is suffering from Gaucher'sdisease.

EXAMPLES

In the Examples described in the following human plasma was used as asample from a subject. Nevertheless a person skilled in the art willacknowledge that depending on the used type of sample from a subject,e.g. comprising saliva, liquor, plasma, serum, full blood, blood on adry blood filter card or another blood product, the method of thepresent invention has to be adjusted to the type of sample andfurthermore a cut-off level has to be determined for each type of sampleaccording to the method described in the following examples. The presentinventors have found that using a sample of human serum in the method asdescribed below instead of a sample of human plasma will lead toidentical results according to a detection of and a thus determinedlevel of free lyso-Gb1, if the sample of human serum and the sample ofhuman plasma derive from the same subject, and were taken at the sametime point; and wherein the samples were measured in parallel; and, moreparticularly, will lead to the same cut-off level. Without whishing itbe bound and in way of illustrative examples, by use of saliva from ahuman patient a method may be adjusted in dependence of a pH value ofthe sample; or a cut-off level may be determined being 20 ng/ml if usingfull blood or blood collected on a dry blood filter card as a samplefrom a subject.

Example 1: Method for the Detection of Free Lyso-Gb1 in Human Serum

Equipment

For detecting free lysoGb-1 in a sample of plasma from a subject thefollowing equipment was used.

Apparatus/Piece of Equipment Type/Producer HPLC pump Series 200, PerkinElmer, USA Sample injector Series 200, Perkin Elmer, USA Column ovenSeries 200, Perkin Elmer, USA Mass selective detector API 4000 Q TRAP,AB SCIEX, USA/Canada Multi-tube vortexer Henry Troemner LLC, USADVX-2500 Vortex mixer Vortex Genie 2; Scientific Industries, USACentrifuge Megafuge 1.0; Heraeus, Germany Multipette(s), pipette(s)Eppendorf, Germany Water bath SW21-C, Julabo, Germany

Reagents

For detecting free lysoGb-1 in a sample of plasma from a subject thefollowing reagents were used.

To that extent that values depend on temperature (e.g. the pH value)such values were determined at a temperature of 25° C.

Reagent Purity Acetonitrile (ACN) HPLC-grade or Gradient grade Acetone99.5% Dimethylsulfoxide (DMSO) HPLC grade Ethanol (EtOH) p.a., 96%Formic acid (FA) p.a., 98-100% Methanol (MeOH) Gradient (LiChrosolv)Trifluoroacetic acid (TFA) purum > 98% Water ASTM-I

The abbreviation “p.a.” as used herein means “pro analysis”.

The term “purum” as used herein, preferably means a commercial grade ofa chemical compound having a purity of the above specified value.

ASTM-I as used herein refers to a water grade standard purity achievedby purification methods comprising Reverse Osmosis and Ultraviolet (UV)Oxidation.

Preparation of Calibration Standards

A Lyso-Gb1 stock solution was prepared dissolving 1.70 mg Lyso-Gb1 (asdelivered by Matreya) in 5 mL of MeOH.

Subsequently the solution V1-A-534 was prepared as a mixture of 12 μL ofLyso-Gb1 stock solution and 5 mL DMSO/MeOH (1:1; v/v) as displayed inthe following:

Label of Volume of volume of resulting exp. conc. solution solventsolution [μg/mL] [μL] solution [mL] solvent V1-A-534 0.79968 12Lyso-Gb1-stock 5 DMSO/MeOH (1:1; v/v)

Subsequently the Calibration Standards were prepared by spiking solutionV1-A-534 or higher concentrated Calibration Standards into the solventMeOH/water (1:1; v/v).

A detailed spiking scheme will be displayed in the following.

Label of Volume of volume of resulting concentration solution solventVolume solution [ng/mL] [μL] solution [mL] solvent [ml] Std9A- 102.12366 V1-A- 2.5 MeOH/water 2.866 534 534 (1:1; v/v) Std8A- 40.970 162V1-A- 3 MeOH/water 3.162 534 534 (1:1; v/v) Std7A- 15.321 353 Std9A- 2MeOH/water 2.353 534 534 (1:1; v/v) Std6A- 6.1464 353 Std8A- 2MeOH/water 2.353 534 534 (1:1; v/v) Std5A- 2.5906 135 Std8A- 2MeOH/water 2.135 534 534 (1:1; v/v) Std4A- 1.0577 53 Std8A- 2 MeOH/water2.053 534 534 (1:1; v/v) Std3A- 0.41004 55 Std7A- 2 MeOH/water 2.055 534534 (1:1; v/v) Std2A- 0.15868 53 Std6A- 2 MeOH/water 2.053 534 534 (1:1;v/v) Std1A- 0.050049 39.4 Std5A- 2 MeOH/water 2.0394 534 534 (1:1; v/v)

For calibration, calibration standards having seven concentration levelsbetween 0.400 and 100 ng/mL were used, namely Calibration StandardsStd3A-534, Std4A-534, Std5A-534, Std6A-534, Std7A-534, Std8A-534 andStd9A-534.

Preparation of Control Samples

Control samples were prepared by spiking solution V1-A-534 or a higherconcentrated control sample into a blank matrix.

A detailed spiking scheme will be displayed in the following.

Label of Volume of volume of resulting concentration solution blankmatrix Volume solution [ng/mL] [μL] solution [mL] [ml] QC-A1- 1.0013173.6 QC-C1- 8.5 8.6736 534 534 QC-B1- 5.0008 944 QC-C1- 8.5 9.444 534534 QC-C1- 50.029 634 V1-A- 9.5 10.134 534 534Blank Matrix

As a blank matrix, human plasma of a healthy subject was used. A personskilled in the art will acknowledge that said plasma from a healthysubject will contain a native level of free lyso-Gb1. Said native levelof free lyso-Gb1 is about 1.4 ng/ml according to the methods of thepresent invention. It is thus obvious that control samples prepared byspiking of the blank matrix, the blank matrix comprising said nativelevel of free lyso-Gb1, also comprise said native level of free lyso-Gb1in addition to the level of free lyso-Gb1 obtained by spiking with aconcentrated solution or higher concentrated control sample.Accordingly, the level of free lyso-Gb1 in the control samples is asfollows:

QC-A1-534 1 ng/mL + native concentration in blank matrix QC-B1-534 5ng/mL + native concentration in blank matrix QC-C1-534 50 ng/mL + nativeconcentration in blank matrix 

A person skilled in the art will acknowledge that human plasma of ahealthy subject used as blank matrix can be purchased at any commercialsource known to the one skilled in the art. It is important to note thatif accidentally plasma of a non-healthy subject, i.e. of a subjecthaving Gaucher's disease, is used as the blank matrix, this will resultin unusually high levels of free lyso-Gb1 in the control samplesdetermined by the method according to the present invention and thuswill be immediately recognized, as the tolerance of the method isdetermined as being within a range of 15% above or below the estimatedlevels of the controls subjected to the method according to the presentinvention.

Study Samples

Preparation of Internal Standard

The Internal Standard (IS1) stock solution was prepared dissolving 1.00mg of Lyso-Gb2 (as delivered by Matreya) in 2 mL of DMSO/MeOH (1/1;vol/vol).

Subsequently the Internal Standard Working Solution was prepared as amixture of 410 μL of IS1 stock solution and 500 mL of ethanol. Theethanol may be purchased from any commercial source, wherein the ethanolis absolute ethanol having a grade suitable for the methods describedherein. A person skilled in the art will recognize that proteinscontained in 50 μl of a sample have to precipitate if 100 μL of saidInternal Standard working solution are added to the sample.

Storing of Samples and Solutions

Control samples or study samples either were immediately stored below−20° C. at once or aliquots were transferred into new glass vials beforestoring under the same conditions.

Concentrated solutions (stock solutions, V1-A-534 etc.) as well asInternal Standard stock solutions were frozen below −20° C. pending nextspiking.

Internal Standard working solutions were stored between 2° C. and 8° C.until use. The present inventors have found that free lyso-Gb1 is stablein the above mentioned solutions. More precisely, the level of freelyso-Gb1 of a plasma and/or a serum sample of a Gaucher's diseasepatient determined by the methods according to the present inventionwere found to be identical, if the level of free lyso-Gb1 was determinedin said samples prior to and after storage at 37° C. for 2 days.Accordingly, the solutions and samples of the present invention can betransported in a number of ways well known to one skilled in the art,wherein the use of a cold chain for transportation of patient materialis preferred but not necessarily required. A person skilled in the artwill also know methods and their respective conditions for appropriatestorage of solutions and samples, wherein, for example, said solutionsand samples may be stored for several weeks.

Sample Preparation for Analysis

All samples used in an analytical batch are prepared for analysis asfollows:

-   -   Frozen samples were thawed at approximately 20 to 25° C. in a        water bath taking from ambient conditions. After thawing the        samples were mixed.    -   50 μL of the sample were transferred into a sample vial. 100 μL        of Internal Standard working solution (in EtOH) was added to the        sample.    -   The thus obtained mixture was subsequently mixed using a        DVX-2500 Multi-tube vortex device at 2500 rpm for about 30        seconds.    -   The thus obtained mixture was centrifuged for phase separation        at 4000 rpm for 2 minutes.    -   Transfer of a volume of the supernatant adequate to injection        purposes (approx. 100 μL) into appropriate (conical)        auto-sampler vials.        Methods        Chromatographic and Auto-Sampler Parameters

The samples prepared for analysis as described above were subsequentlysubjected to the method described in the following:

Parameter Scheduled range/description Mobile phase solvent A 50 mM FA inwater Mobile phase solvent B 50 mM FA in ACN/acetone (1:1; vol/vol)Chromatographic run 0.0-4.0 min linear gradient: 5% B → 66% B 4.1-5.1min isocratic: 100% B 5.1-5.9 min isocratic: 5% B Flow 0.9 mL/minInjection volume 5 μL Injector flush 0.1% TFA in 70% MeOH Column +Precolumn ACE 3 C8, 50 × 2.1 mm ID + Security Guard C8 Columntemperature 60° C. Retention time approx. 3.4 to 3.6 min: lyso-Gb1 andlyso-Gb 2 (IS)

The ACE 3 C8 column (ACE C8 column Nr. ACE-112-0502) used herein hasbeen purchased from Advanced Chromatography Technologies, Aberdeen.

It will be appreciated by a person skilled in the art that parameterswhere a “+” range is indicated represent parameters which may beadjusted between sequences. A sequence as used herein, preferably is abatch of defined numbers of samples, preferably 250 in maximum analyzedsequentially, wherein parameters comprising flow and temperature remainunchanged. Adjustments and calibrations performed between sequences areknown to those skilled in the art and comprise exchange of the column.

These adjustments within the specified limits are minor changes and arerecorded within the raw data of the study at the measuring station.

Detection

The thus prepared samples were subsequently subjected to the detectionmethod the parameters of which are described in the following:

MS Ionisation mode: Electrospray Ionisation (ESI) MS polarity: positiveMS detection mode: Multiple reaction monitoring (MRM) Vaporizertemperature: 500° C. ± 50° C. Ionisation voltage: 5.5 kV Collisionallyactivated low dissociation (CAD) gas: Gas 1: Pressure = 45 psi Gas 2:Pressure = 60 psi Curtain gas: pressure = 40 psi Lateral position: 5units Vertical position: 4 units Quadrupole resolution unit → unitTransitions 462.4 → 282.2 m/z lyso-Gb1 624.5 → 282.2 m/z lyso-Gb2(Internal Standard) DP (declustering potential) 40 V CXP (collision cellexit potential) 8 V

A person skilled in the art will acknowledge that methods for detectingfree lyso-Gb1 and/or determining the level of free lyso-Gb1 in a samplefrom a subject using mass spectrometric analysis may also employ othertransitions and fragments which allow for specific detection of and/orquantification of free lyso-Gb1 in said sample from a subject.

Evaluation and Calculation of Results

To evaluate and to calculate results obtained with the above specifiedmethods the following protocol were applied.

Rounding Procedure

Concentration data fed into and retrieved from the chromatographic datasystem (CDS) were rounded to five significant digits. Furthercalculations in the spreadsheet were performed to full computationalaccuracy and subsequently rounded to the significant digits/decimalplaces to be reported. Hence, deviations of intermediate results mightoccur caused by rounding. Accuracy and coefficients of variation (CV)will be reported with one and two decimal places, respectively.

Note referring to the rounding procedure: The last digit reported wouldbe up-rounded if the subsequent digit was equal or greater than “5”.

Regression and Statistics

Based on Calibration Standards the calibration curve fitting wereestablished using the data processing software by means of peak arearatios (peak area of free lyso-substance contained in the sample fromthe subject/peak area of Internal Standard). Free lyso-substanceconcentrations were evaluated using an Internal Standard methodAquadratic (y=ax²+bx+c) regression model using the weighting factor1/conc. will be used to calculate the concentration of each analyte inevery batch to be evaluated. The concentrations were calculated by meansof the following formula:

${concentration} = \frac{{- b} \pm \sqrt{b^{2} - {4{a\left( {c - {{peak}\mspace{14mu}{area}\mspace{14mu}{ratio}}} \right)}}}}{2a}$

Based thereon mean values, precision results (in terms of CVs) andaccuracies (formula shown below) will be calculated using the program“Lotus 123”.

${{accuracy}\mspace{14mu}(\%)} = {\frac{{calculated}\mspace{14mu}{concentration}}{{expected}\mspace{14mu}{concentration}} \cdot 100}$

Appropriate statistical models are described in e.g.

-   -   Green, J. R., Statistical Treatment of Experimental Data        (Elsevier, New York, 1977), page 210 ff.    -   Lothar Sachs, Angewandte Statistik—Anwendung statistischer        Methoden (Springer, Berlin, Heidelberg, N.Y., Tokyo 1984)        Software

Data acquisition, data processing, statistics and calculations wereperformed using Analyst® software 1.4.2 or higher (AB SCIEX, USA/Canada)as well as Lotus 1-2-3 97 or higher (Lotus Corp, USA).

Handbooks

Handbook Arbeiten mit SmartSuite 97 (Lotus Development Corp., 1997)Documentation of Documentation of Analyst ® Software (AB SCIEX, softwareused USA/Canada): Operator's Manual & Operator's Manual Addendum “NewFunctionality in Analyst 1.2” and Online Help System Analyst 1.4 (orhigher)

Example 2: Genetic Testing and Classification of Study Participants

After consenting of patients to participation in the study, patientswere subjected to a genetic testing for mutations of theglucocerebrosidase gene. Accordingly, 5 to 10 ml of EDTA blood weresequenced according to Seeman et al. (Seeman et al., 1995). Wereappropriate other genes beside the glucocerebrosidase gene weresequenced in addition, particularly in controls. Furthermore thechitotriosidase gene was sequenced for detection of the 24 bpduplication as mentioned above. Said genetic testing was controlledusing test samples of age and sex matched control patients.

253 subjects were tested.

According to the result of the above described genetic testing, patientsparticipating in the study were classified into the following groups:

1.) Patients having Gaucher's disease: gold standard for the diagnosiswas the detection of two pathogenic mutations within theglucocerebrosidase gene, either homozygous or compound heterozygous(group is named in the figures as “Gaucher”);

2.) Patients being heterozygous carriers of one mutation within theglucocerebrosidase gene (typically relatives of affected patients)(group is named in the figures as “heterozygote”)

3.) patients with other lysosomal storage disorders as control (group isnamed in the figures as “other LSD”); this comprises patients withsphingomyelinase deficiency (Niemann Pick A/B), Krabbe disease andNiemann Pick C1; all diagnoses have been proven by the detection of twopathogenic mutations4.) healthy age and gender matched controls (group is named in thefigures as “control”) The following table 1a shows the classifying ofpatients into the above described groups according to the results of theabove described genetic testing.

TABLE 1a Subjects classified by results of genetic analysis cases validmissing total Groups (Dgn) N percentage N percentage N percentagecontrol 140 100.0% 0 0% 140 100.0% Heterozygous 13 100.0% 0 0% 13 100.0%(carrier) Gaucher 59 100.0% 0 0% 59 100.0% other LSD 20 100.0% 0 0% 20100.0%

The distribution of the gender of the 232 German patients as well as thedistribution of the gender of 21 Israeli patients are depicted in Table1 b.

TABLE 1b 232 German subjects and 21 Israeli classified by gender GermansIsrael N % N % total 232 21 Sex male 146 57.0 11 52.4 female 110 43.0 1047.6

The following table 1c shows the distribution of the age of the 232German patients and the classification of said patients based on theresults of the above described genetic testing as well as the gender ofsaid patients.

TABLE 1c Patient characteristics of 253 subjects Healthy Heterozygouscontrols carrier Gaucher Other LSD N subjects 140 13 80 20 N samples 15515 287 28 Age in years 28.5 35.0 30.0 23.5 (median, (4.8-47.3)(30.5-58.5) (8.0-48.0) (4.0-43.5) interquartile (n = 134) (n = 13) (n =79) (n = 14) range) (number of cases) male female male female malefemale male female n 79 61 8 5 45 35 12 8 Age (median, 25.5 34.0 33.539.0 22.0 32.5 21.0 34 interquartile (5.3-47.0) (3.8-48.8) (26.0-51.8)(33.0-69.5) (7.5-50.0) (12.8-43.3) (3.3-30.3) (8.8-45.8) range)

The level of free lyso-Gb1 in samples of said 253 subjects wasdetermined according to the method described in Example 1. The level offree lyso-Gb1 in samples from said patients depending on theclassification by genetic analysis is shown in FIG. 1A. FIG. 1B showsthe level of free lyso-Gb1 in samples from said patients depending onthe classification based on the genetic analyses and on the gender ofthe patients.

The type of mutation and the distribution of the types of mutations ofthe glucocerebrosidase gene in patients classified as Gaucher's diseasepatients according to the results obtained in the genetic testing asdescribed above are depicted in Table 2 below.

TABLE 2 Distribution of mutations being detected in the German Gaucherpopulation (166 alleles) type of mutation n % N370S 54 32.5% L444P 3319.9% RecNciI 15 9.0% G202R 4 2.4% D409H 3 1.8% Rec 3 1.8% G355A 2 1.2%IVS2+1A>G 2 1.2% L335V 2 1.2% L444R 2 1.2% R120W 2 1.2% R285H 2 1.2%RecAP2 2 1.2% T226I 2 1.2% T231R 2 1.2% T491I 2 1.2% V398L 2 1.2%A46term 1 0.6% A495P 1 0.6% A88P 1 0.6% C287F 1 0.6% F216Y 1 0.6% G82A 10.6% H255Q 1 0.6% I93F 1 0.6% IVS3+1G>A 1 0.6% L324Q 1 0.6% N234S 1 0.6%N409S 1 0.6% P161R 1 0.6% P178S 1 0.6% P29X 1 0.6% P68fs 1 0.6% Q326K 10.6% R120Q 1 0.6% R257ter 1 0.6% R359Q 1 0.6% R502C 1 0.6% R502H 1 0.6%RecAF3 1 0.6% RecAF4 1 0.6% RecAH3 1 0.6% RecTL 1 0.6% S13L 1 0.6% S146L1 0.6% S237F 1 0.6% S364N 1 0.6% V398L 1 0.6% W184R 1 0.6%Measurement of Chitotriosidase Activity

Chitotriosidase activity was measured as essentially described in Hollaket al. (Hollak C E, van Weely S, van Oers M H, Aerts J M. Markedelevation of plasma chitotriosidase activity. A novel hallmark ofGaucher disease. J Clin Invest. 1994 March; 93(3):1288-92) by incubating10 μl of EDTA plasma or serum with 100 μl of 0.022 mM fluorogenicsubstrate 4-methylumbelliferyl-fl-D-NN,N′-triacetylchitotriose (4MU-chitotrioside; Sigma Aldrich, ST. Louis, Mo., USA) as substrate inMcIlvain buffer (0.1 M citric acid/0.2 M sodium phosphate, pH 5.2) at37° C. In Gaucher's disease patients, samples were diluted 50× indemineralized water before incubation. After 30 min the reaction wasstopped with 200 μl of 0.5 M glycine/NaOH buffer (pH 10.5) by mixing atroom temperature. The substrate hydrolysis by chitotriosidase producesthe fluorescent molecule 4-methylumbelliferone, which was quantifiedwith a fluorimeter (Tecan Group Ltd., Männedorf, Switzerland),excitation at 366 nm and emission at 446 nm, and compared with astandard 4-methylumbelliferone calibration curve. Chitotriosidaseactivity was expressed as nanomoles of substrate hydrolyzed per hour permilliliter of incubated serum.

Quantification of CCL18

CCL18 in plasma was quantified with a DuoSet ELISA Development kitpurchased from R&D Systems, Minneapolis, Minn., USA in accordance withthe manufacturer's instructions. The sensitivity of the method was 5pg/ml.

Example 3: Diagnosis of Gaucher's Disease Using Free Lyso-Gb1 as aBiomarker

The protocols described in Example 1 above were used to generateHPLC-mass spectrometric chromatograms of 485 blood samples derived fromthe 253 subjects. Exemplary HPLC-mass spectrometric chromatogramsdisplaying peak intensity of free lyso-Gb1 and IS of three samples fromtwo Gaucher's disease patients and one healthy control person aredepicted in FIG. 5A, FIG. 5B and FIG. 5C.

Gold standard for the classification of patients into the group“Gaucher”, was based on the sequencing of the entire coding area as wellas the the intron-exon-boundaries of the glucocerebrosidase geneaccording to the genetic testing as described in Example 2 resulting inthe detection of either a homozygous mutation or a compoundheterozygosity.

The results of a determination of the levels of Chitotriosidase or CCL18in samples from patients were available in 58 or 44 Gaucher's diseasepatients, respectively. Said results were obtained as described inExample 2.

For comparing the diagnostic value of the different biomarkers and forthe calculation of correlations between the biomarkers the data obtainedby the method described above was first aggregated by using the earliestmeasured level of every marker for Gaucher's disease patients beforetherapy and the highest level for non-Gauchers for a particular patientif more than one blood sample was available.

Paired sample statistical techniques were used for the comparison of twobiomarkers. The method exploits the mathematical equivalence of the AUCto the Mann-Whitney U-statistic (Delong E. R., Delong D. M.,Clarke-Pearson D. L. (1988) Comparing the areas under two or morecorrelated receiver operating characteristic curves: a nonparametricapproach, Biometrics, 44, 837-45.).

The accuracy of levels of the different biomarkers (free lyso-Gb1,Chitotriosidase and CCL18) obtained by the method described in Example 1above was evaluated to discriminate patients with Gaucher's disease frompatients without having Gaucher's disease using Receiver OperatingCharacteristic (ROC) curve analysis (Metz C.E. (1978) Basic principlesof ROC analysis, Semin Nucl Med, 8, 283-98; Zweig M. H., Campbell G.(1993) Receiver-operating characteristic (ROC) plots: a fundamentalevaluation tool in clinical medicine, Clin Chem, 39, 561-77).Measurement of Chitotriosidase activity and CCL18 was performed asdescribed in Example 2 herein.

The ROC curves were calculated using PASW Statistics 18, Release Version18.0.2 (©SPSS, Inc., 2009, Chicago, Ill., www.spss.com). The comparisonsof ROC curves and the linear mixed models were done using SAS software,Version 9.2 of the SAS System for Windows. (©, 2008 SAS Institute Inc.,Cary, N.C., USA).

The ROC-curves comparing accuracy of levels of chitotriosidase and freelyso-Gb1 are shown in FIG. 2A and the ROC-curves comparing accuracy oflevels of CCL18 and free lyso-Gb1 are shown in FIG. 2B, respectively.

The results depicted in the ROC-curves shown in FIG. 2A and FIG. 2B alsoshow the specificity and the sensitivity of the method according to thepresent invention depending on different cut-off levels of freelyso-Gb1. Table 3 below shows accordingly the Sensitivity and theSpecificity of the method according to the present invention dependingon different cut-off levels of free lyso-Gb1.

TABLE 3 Sensitivity and Specificity of the method for diagnosingGaucher's disease depending on the cut-off level of free lyso-Gb1 in theGerman subjects (n = 232) Cut-off level >2.8 [ng/mL] >4.1 [ng/mL] >5[ng/mL] Sensitivity 100.0% 100.0% 100.0% Specificity 97.7% 99.4% 100.0%

Comparing the level of the biomarker in a sample from a subjectdetermined by the method according to the present invention to a cut-offlevel, preferably a cut-off level allowing for a diagnosis having highspecificity and high sensitivity thus allows for diagnosing Gaucher'sdisease in said subject, wherein an elevated level of the biomarker inthe sample from the subject compared to the cut-off level is indicativefor the subject for suffering from or for being at risk for developingGaucher's disease and wherein a lower level of the biomarker in thesample from the subject compared to the cut-off level is indicative forthe subject for not suffering from or for not being at risk fordeveloping Gaucher's disease.

The area under the curve (AUC) and the 95% confidence limits for thedifferent biomarkers are reported in table 4.

TABLE 4 Sensitivity and specificity for different biomarkers with regardto diagnose Gaucher. Chitotriosidase CCL18 free lyso-Gb1 (n = 228/58 (n= 210/44 (n = 232/59 Gaucher) Gaucher) Gaucher) Cut-off level >145[nmolMU/h/ml] >166 [ng/ml] >5 [ng/mL] Sensitivity 93.1% 79.5% 100.0%Specificity 90.0% 79.5% 100.0% AUC and 95% CI in 0.96 (0.92-1.00) 0.87(0.80-0.93) 1.00 (1.00-1.00) ROC Analysis

Accordingly, in table 3 the sensitivity and the specificity of thedepicted biomarkers used in a method for diagnosing Gaucher's disease ina sample from a subject is compared using a cut-off level having thehighest AUC in the respective method using the respective biomarker.Depicted is the ideal cut-off level of the respective method.Measurement of Chitotriosidase activity and CCL18 was performed asdescribed in Example 2 herein. Free lyso-Gb1 was determined according tothe method of the present invention. The ideal cut-off level is 5 ng/ml.

A person skilled in the art will acknowledge that the method accordingto the present invention using free lyso-Gb1 as a biomarker fordiagnosing Gaucher's disease is clearly advantageous over methods usingCCL18 or Chitotriosidase. This is especially true since at least 6% ofthe Caucasian population and up to 35% e.g. of the Latin Americanpopulation, including those with Gaucher's disease, are deficient inchitotriosidase activity.

Accordingly, levels of free lyso-Gb1 determined in a sample from asubject according to the method of the instant application higher than5.0 ng/mL allow for diagnosing that the subject is suffering from or isat risk for developing Gaucher's disease with a sensitivity of and witha specificity of 100%.

Example 4: Analysis of Change of Biomarkers Over Time

The method and patients used in connection with this Example were thoseas described in Examples 1 to 3.

For analyzing how the level of biomarkers changed over time in patientshaving Gaucher's disease non aggregated data was analyzed for thosepatients for whom more than one blood sample was analyzed. A time pointzero was set to the first measure under therapy for every patient.

The levels of free lyso-Gb1 over time for individual patients are shownin FIG. 3A, FIGS. 3B and 3C.

To test for the significance of a time dependent reduction of freelyso-Gb1 levels indicative for a successful therapy, free lyso-Gb1levels after start of a therapy were compared to free lyso-Gb1 levelsbefore start of a therapy using linear mixed models. Untreated patientsdemonstrate no significant reduction of free lyso-Gb1 over the time.

Therefore the values of free lyso-Gb1 levels were logarithmised toovercome the skewness in the distribution of the values. To account forthe heterogeneity between patients in the starting values as well as inthe rate of change random intercept and slope models were used. In allmodels the observed heterogeneity was statistically significant. Onlyp-values for the linear time reduction are reported.

The values for time and those values which incorporated a squared termfor time were centered to test for a curvilinear relation between timeand marker level for Chitotriosidase and for CCL18. For free lyso-Gb1the squared term did not improve the model and was not incorporated inthe final model.

As a therapy German patients have been treated with 40 U/kg body weightin the mean, wherein units refers to units of recombinantglucocerebrosidase in ERT. The reduction in free lyso-Gb1 isspecifically intense after start of therapy (after 6 months<0.0001). Butalso the reduction over time is significant (<0.0001). There is areduction of free lyso-Gb1 after 12 months of treatment in a range of60% in the mean.

The features of the present invention disclosed in the specification,the claims, the sequence listing and/or the drawings may both separatelyand in any combination thereof be material for realizing the inventionin various forms thereof.

The invention claimed is:
 1. A method for generating quantitative datafor a subject comprising the steps of: a) determining the level of abiomarker in a subject having Gaucher's disease; b) determining againthe level of the biomarker in a sample from said subject afteradministration of a therapy to the subject: wherein the sample is ablood sample; and wherein the biomarker is free lyso-Gb1.
 2. The methodof claim 1, wherein the therapy is selected from the group consisting ofenzyme replacement therapy (ERT), substrate replacement therapy (SRT)and a chaperone based therapy.
 3. The method of claim 1, furthercomprising detecting at least one additional biomarker in the samplefrom the subject.
 4. The method of claim 3, wherein the at least oneadditional biomarker is selected from the group comprisingChitotriosidase and CCL18.
 5. The method of claim 1, wherein thebiomarker is detected by means of immunoassay, mass spectrometricanalysis, biochip array, functional nucleic acids and/or a fluorescentderivative of free lyso-Gb1.
 6. The method of claim 1, wherein thebiomarker is detected by means of mass spectrometric analysis.
 7. Themethod of claim 1, wherein a dosage of the therapy is to be increased ifthe level of the biomarker in the sample as determined in step b) ishigher than the level of the biomarker in the sample as determined instep a).
 8. The method claim 1, wherein the sample is blood on a dryblood filter card.